Feline IL-18 nucleic acid molecules

ABSTRACT

The present invention relates to canine and feline proteins. In particular, the present invention discloses feline interleukin-18, feline caspase-1, feline interleukin-12 single chain and canine interleukin-12 single chain proteins. The present invention also includes feline interleukin-18, feline caspase-1, feline interleukin-12 single chain and canine interleukin-12 single chain nucleic acid molecules encoding such proteins, antibodies raised against such proteins and/or inhibitors of such proteins or nucleic acid molecules. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to evaluate and regulate an immune response in an animal.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/821,670, filed Apr. 9, 2004, entitled “FELINE IL-12 SINGLECHAIN NUCLEIC ACID MOLECULES”; which is a divisional of U.S. patentapplication Ser. No. 09/917,265, filed Jul. 27, 2001, now issued as U.S.Pat. No. 6,818,444 B2, entitled “CANINE AND FELINE PROTEINS, NUCLEICACID MOLECULES AND USES THEREOF”; which claims priority to U.S.Provisional Application Ser. No. 60/223,016, filed Aug. 4, 2000,entitled “CANINE AND FELINE PROTEINS, NUCLEIC ACID MOLECULES AND USESTHEREOF”.

FIELD OF THE INVENTION

The present invention relates to canine and feline proteins. Inparticular, the present invention relates to feline interleukin-18(IL-18), feline caspase-1 (casp-1), feline interleukin-12 (IL-12) singlechain, and canine interleukin-12 (IL-12) single chain proteins andincludes nucleic acid molecules encoding such proteins, antibodiesraised against such proteins and/or inhibitors of such proteins ornucleic acid molecules. The present invention also includes therapeuticcompositions comprising such nucleic acid molecules, proteins,antibodies and/or inhibitors, as well as their use to evaluate andregulate an immune response in an animal.

BACKGROUND OF THE INVENTION

Regulation of immune and inflammatory responses in animals is importantin disease management. Immune responses can be regulated by modifyingthe activity of immunoregulatory molecules and immune cells. Examples ofsuch immunoregulatory molecules include IL-18, caspase-1 and IL-12.These molecules have been found to play a role in the treatment ofseveral disorders including allergy, cancer, and pathogenic infection.

Monocytes and macrophages represent the first line of defense againstdisease. Various diseases and infections activate transcriptional andposttranslational events in monocytes and macrophages, which lead to theproduction of cytokines such as IL-18 and IL-12. These cytokines in turnactivate responses in T and B cells helping to eliminate pathogensand/or disease in a animal. Both IL-18 and IL-12 augment cellularimmunity by stimulating T cells to produce interferon gamma (IFN-γ)which inhibits the production of IgE formation without compromising Bcell proliferation. IL-18, formerly referred to as interferon gammainducing factor (IGIF), stimulates T cells to produce IFN-γ and has beenisolated from humans, dogs, and mice. A cDNA encoding human IL-18 wasisolated and used to express recombinant human IL-18 by Ushio et al.,1996, J. Immunol. 156, 4274-4279, GenBank accession number D49950.Feline IL-18 cDNA has a 85.8% homology to human IL-18 cDNA and felineIL-18 protein has a 81.7% homology to human IL-18 protein. A cDNAencoding canine IL-18 was isolated and used to express recombinantcanine IL-18 by Okano et al., 1999, J. Interferon Cytokine Res. 19,27-32, GenBank accession number Y11133. Feline IL-18 cDNA has a 90.7%homology to canine IL-18 cDNA and feline IL-18 protein has a 88.5%homology to canine IL-18 protein. A cDNA encoding murine IL-18 wasisolated and used to express recombinant murine IL-18 by Okamura et al.,1995, Nature 378, 88-91, GenBank accession number D49949. Feline IL-18cDNA has a 73.8% homology to murine IL-18 cDNA and feline IL-18 proteinhas a 70% homology to murine IL-18 protein. A cDNA encoding rat IL-18was isolated by Culhane, et al. Mol. Psych. 3, 362-366 (1998), GenBankaccession number AJ222813. Feline IL-18 cDNA has a 73.4% homology withrat IL-18 cDNA, and feline IL-18 protein has a 70.7% homology with ratIL-18 protein. A cDNA encoding equine IL-18 was isolated by Nicolson, etal. (unpublished, direct submission to GenBank, accession numberY11131). Feline IL-18 cDNA has a 92% homology to equine IL-18 cDNA andfeline IL-18 protein has a 89% homology to equine IL-18 protein. A cDNAencoding pig IL-18 was isolated by Penha-Goncalves, et al. (unpublished,direct submission to GenBank, accession number Y11132. Feline IL-18 cDNAhas a 90.2% homology to pig IL-18 cDNA and feline IL-18 protein has a85.9% homology to pig IL-18 protein. Expression of active IL-18 iscontrolled by caspase-1 (IL-1β converting enzyme). That is, IL-18 lacksa signal peptide so the precursor form of IL-18 (pro IL-18) is cleavedby caspase-1 resulting in a mature protein that is biologically active.

IL-12 is a heterodimer comprised of two subunits p40 and p35 which arecovalently linked by a disulfide bond to form an active molecule.Simultaneous expression of the two subunits is necessary for theproduction of the biologically active heterodimer. Both human and murinep35 and p40 IL-12 single chain proteins (i.e., a single proteincontaining both p35 and p40 subunits) have been produced; see e.g.,Lieschke et al., 1997, Nature Biotechnology 15, 35-40. Co-expression ofthe human and murine p35 and p40 cDNA subunits of IL-12 resulting in abiologically active IL-12 heterodimer was achieved by Gubler et al.,1991, Proc. Natl. Acad. Sci. U.S.A., 88, 4143-4147 and Schoenhaut etal., 1992, J. Immunol., 148, 3433-3440, respectively. cDNAs encodingcanine IL-12 p35 and p40 subunits were isolated and co-transfected toexpress canine IL-12 by Okano et al., 1997, J. Interferon Cytokine Res.17, 713-718. cDNAs encoding feline p35 and p40 subunits have beenisolated and expressed; see, for example, Fehr et al., 1997, DNA Seq. 8,77-82; Schijns et al., 1997, Immunogenetics 45, 462-463; Bush et al.,1994, Molec. Immunol. 31, 1373-1374. At the amino acid level, canine andfeline IL-12 p40 subunit share 92.7 percent identity to each other;share 84.8 and 84.2 percent identity to human IL-12 p40, respectively;and share 67.4 and 68.9 percent identity to murine IL-12 p40,respectively. IL-12 shares some biological activities with IL-18including IFN-γ production in T cells. IL-18 and IL-12 in combinationwork synergistically to increase IFN-γ production in T cells; as suchthese cytokines when utilized alone or in combination can be veryeffective in mediating IgE responses.

Caspase-1 may play a key role in the processing of IL-18 precursor incells where IL-18 is produced. It may be that coexpression of caspase-1along with IL-18 may be necessary for the proper processing of the IL-18precursor and enhanced secretion of the processed IL-18 maturepolypeptide. A cDNA encoding equine caspase-1 was isolated by Wardlow,et al. (unpublished; direct submission to GenBank, accession numberAF090119). Feline caspase-1 cDNA has a 71% homology to equine caspase-1cDNA and feline caspase-1 protein has a 48.8% homology to equinecaspase-1 protein. A cDNA encoding equine caspase-1 was isolated byCerretti, et al. (Science 256, p 97-100 (1992); GenBank accession numberM87507). Feline caspase-1 cDNA has a 60% homology to human caspase-1cDNA and feline caspase-1 protein has a 60% homology to human caspase-1protein. A cDNA encoding rat caspase-1 (called interleukin-1 betaconverting enzyme) was isolated by Keane, et al. (Cytokine 7(2) 105-1101995); GenBank accession number U14647). Feline caspase-1 cDNA has a55.4% homology to rat caspase-1 cDNA and feline caspase-1 protein has a40.2% homology to rat caspase-1 protein. A cDNA encoding murinecaspase-1 was isolated by Molineaux, et al. (Proc Natl. Acad. Sci 90,1809-1813, 1993); GenBank accession number L28095). Feline caspase-1cDNA has a 55.7% homology to murine caspase-1 cDNA and feline caspase-1protein has a 38.5% homology to murine caspase-1 protein. A cDNAencoding canine caspase-1 was isolated by Taylor, et al. (2000) DNA Seq.10(6), pp 387-394; GenBank accession number AF135967). Feline caspase-1cDNA has a 90% homology to canine caspase-1 cDNA.

To date, however, neither IL-18 nor caspase-1, nor the nucleic acidmolecules encoding such proteins, have been isolated from cats. Neitherhave IL-12 single chain proteins been produced using feline or canineIL-12 subunits. As such there remains a need for compounds and methodsto regulate an immune response in cats and dogs through manipulation ofIL-18, caspase-1 and IL-12 single chain activities.

SUMMARY OF THE INVENTION

The present invention relates to canine and feline proteins, nucleicacid molecules encoding such proteins, antibodies raised against suchproteins and/or inhibitors of such proteins or nucleic acid molecules.In a preferred embodiment, the present invention relates to felineinterleukin-18 (IL-18), feline caspase-1 (casp-1), feline interleukin-12(IL-12) single chain and canine interleukin-12 single chain proteins,nucleic acid molecules, antibodies and inhibitors. The present inventionalso includes therapeutic compositions comprising such nucleic acidmolecules, proteins, antibodies and/or inhibitors, as well as their useto evaluate and regulate an immune response in an animal.

One embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (a) an isolated nucleicacid molecule selected from the group consisting of (i) a nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ ID NO:11, and SEQ IDNO:13; and (ii) a nucleic acid molecule comprising at least 70contiguous nucleotides identical in sequence to at least 70 contiguousnucleotides of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ IDNO:41; (b) an isolated nucleic acid molecule selected from the groupconsisting of (i) a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:23 and SEQ ID NO:25, and (ii) a nucleic acid molecule comprising atleast 70 contiguous nucleotides identical in sequence to at least 70contiguous nucleotides of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:25;{circle around (C)}) an isolated nucleic acid molecule selected from thegroup consisting of: (i) a nucleic acid molecule comprising ((a)) anisolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:26, SEQ ID NO:29, and anucleic acid sequence comprising at least 44 contiguous nucleotidesidentical in sequence to at least 44 contiguous nucleotides of a nucleicacid sequence selected from the group consisting of SEQ ID NO:26 and SEQID NO:29; ((b)) a nucleic acid linker of (XXX)_(n) wherein n=0 to 60;and ({circle around (C)})) an isolated nucleic acid molecule comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNO:32, SEQ ID NO:35, and a nucleic acid molecule comprising at least 44contiguous nucleotides identical in sequence to at least 44 contiguousnucleotides of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:32 and SEQ ID NO:35, such that said nucleic acidmolecule of (i) encodes a feline IL-12 single chain protein; and (ii) anucleic acid molecule comprising a nucleic acid sequence fullycomplementary to the coding strand of any of said nucleic acid moleculesas set forth in (i); (d) an isolated nucleic acid molecule selected fromthe group consisting of: (i) a nucleic acid molecule comprising ((a)) anisolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:52 and SEQ ID NO:58, anda nucleic acid sequence comprising at least 47 contiguous nucleotidesidentical in sequence to at least 47 contiguous nucleotides of a nucleicacid sequence selected from the group consisting of SEQ ID NO:46 and SEQID NO:49; ((b)) a nucleic acid linker of (XXX)_(n) wherein n=0 to 60;and ({circle around (C)})) an isolated nucleic acid molecule comprisinga nucleic acid sequence selected from the group consisting of SEQ IDNO:46, SEQ ID NO:49, and a nucleic acid molecule comprising at least 47-contiguous nucleotides identical in sequence to at least 47 contiguousnucleotides of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:46 and SEQ ID NO:49, such that said nucleic acidmolecule of (i) encodes a canine IL-12 single chain protein; and (ii) anucleic acid molecule comprising a nucleic acid sequence fullycomplementary to the coding strand of any of said nucleic acid moleculesas set forth in (i); (e) an isolated nucleic acid molecule selected fromthe group consisting of: (i) a nucleic acid molecule having a nucleicacid sequence that is at least 92 percent identical to a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9,SEQ ID NO:41, SEQ ID NO:11, and SEQ ID NO:13; and (ii) a nucleic acidmolecule comprising a fragment of a nucleic acid molecule of (i) whereinsaid fragment is at least 80 nucleotides in length; (f) an isolatednucleic acid molecule selected from the group consisting of (i) anucleic acid molecule having a nucleic acid sequence that is at least 85percent identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:25, and (ii) anucleic acid molecule comprising a fragment of a nucleic acid moleculeof (i) wherein said fragment is at least 85 nucleotides in length; (g)an isolated nucleic acid molecule selected from the group consisting of:(i) a nucleic acid molecule comprising ((a)) a nucleic acid moleculecomprising a nucleic acid sequence that is at least 87 percent identicalto a nucleic acid sequence selected from the group consisting of SEQ IDNO:26 and SEQ ID NO:29, or a fragment thereof of at least 55 nucleotidesin length; ((b)) a nucleic acid linker of (XXX)_(n) wherein n=0 to 60;and ({circle around (C)})) nucleic acid molecule comprising a nucleicacid sequence that is at least 87 percent identical to a nucleic acidsequence selected from the group consisting of SEQ ID NO:32 and SEQ IDNO:35, or a fragment thereof of at least 55 nucleotides in length, suchthat said nucleic acid molecule (i) encodes a feline IL-12 single chainprotein; and (ii) a nucleic acid molecule comprising a nucleic acidsequence fully complementary to the coding strand of a nucleic acidmolecule as set forth in (i); and (h) an isolated nucleic acid moleculeselected from the group consisting of: (i) a nucleic acid moleculecomprising ((a)) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:52 and SEQID NO:58, and a nucleic acid sequence comprising at least 55 contiguousnucleotides identical in sequence to at least 55 contiguous nucleotidesof a nucleic acid sequence selected from the group consisting of NO:52and SEQ ID NO:58; ((b)) a nucleic acid linker of (XXX)_(n) wherein n=0to 60; and ({circle around (C)})) an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:46, SEQ ID NO:49, and a nucleic acid molecule comprising atleast 55 contiguous nucleotides identical in sequence to at least 55contiguous nucleotides of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:32 and SEQ ID NO:35, such that saidnucleic acid molecule of (i) encodes a canine IL-12 single chainprotein; and (ii) a nucleic acid molecule comprising a nucleic acidsequence fully The present invention also includes recombinantmolecules, recombinant viruses and recombinant cells comprising suchIL-18, caspase-1, and IL-12 single chain nucleic acid molecules andmethods to produce such nucleic acid molecules, recombinant molecules,recombinant viruses and recombinant cells.

Another embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (a) a nucleic acidmolecule having a nucleic acid sequence encoding an IL-18 proteinselected from the group consisting of: (i) a protein selected from thegroup consisting of ((a)) a protein having an amino acid sequence thatis at least 92 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ IDNO:12, and ((b)) a protein comprising a fragment of a protein of ((a)),wherein said fragment is at least 30 amino acids in length; and (ii) aprotein comprising at least 25 contiguous amino acids identical insequence to at least 25 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8 and SEQ ID NO:12; (b) a nucleic acid molecule having a nucleic acidsequence encoding a caspase-1 protein selected from the group consistingof: (i) a protein selected from the group consisting of ((a)) a proteinhaving an amino acid sequence that is at least 85 percent identical toan amino acid sequence selected from the group consisting of SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, and ((b)) a proteincomprising a fragment of a protein of ((a)), wherein said fragment is atleast 30 amino acids in length; and (ii) a protein comprising at least25 contiguous amino acids identical in sequence to at least 25contiguous amino acids selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24; {circle around (C)}))a nucleic acid molecule having a nucleic acid sequence encoding an IL-12single chain protein comprising an IL-12 p40 subunit domain linked to aIL-12 p35 subunit domain, wherein said p40 subunit domain is selectedfrom the group consisting of (i) a p40 subunit protein having an aminoacid sequence that is at least 84 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:27 and SEQ IDNO:30, (ii) a p40 subunit protein comprising a fragment of a protein of(i), wherein said fragment is at least 30 amino acids in length, and(iii) a p40 subunit protein comprising at least 23 contiguous aminoacids identical in sequence to at least 23 contiguous amino acids of anamino acid sequence selected from the group consisting of SEQ ID NO:27and SEQ ID NO:30 and wherein said p35 subunit domain is selected fromthe group consisting of (i) a p35 subunit protein having an amino acidsequence that is at least 84 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36,(ii) a p35 subunit protein comprising a fragment of a protein of (i),wherein said fragment is at least 30 amino acids in length, and (iii) ap35 subunit protein comprising at least 23 contiguous amino acidsidentical in sequence to at least 23 contiguous amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:33 and SEQID NO:36; (d) a nucleic acid molecule having a nucleic acid sequenceencoding an IL-12 single chain protein comprising an IL-12 p40 subunitdomain linked to a IL-12 p35 subunit domain, wherein said p40 subunitdomain is selected from the group consisting of (i) a p40 subunitprotein having an amino acid sequence that is at least 84 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:53 and SEQ ID NO:59, (ii) a p40 subunit protein comprisinga fragment of a protein of (i), wherein said fragment is at least 40amino acids in length, and (iii) a p40 subunit protein comprising atleast 31 contiguous amino acids identical in sequence to at least 31contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:53 and SEQ ID NO:59, and wherein said p35subunit domain is selected from the group consisting of (i) a p35subunit protein having an amino acid sequence that is at least 84percent identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35 subunit proteincomprising a fragment of a protein of (i), wherein said fragment is atleast 40 amino acids in length, and (iii) a p35 subunit proteincomprising at least 31 contiguous amino acids identical in sequence toat least 31 contiguous amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:47 and SEQ ID NO:50; (e) anucleic acid molecule comprising a nucleic acid sequence fullycomplementary to the coding strand of any of said nucleic acid moleculesas set forth in (a), (b), {circle around (C)}), or (d). The presentinvention also includes recombinant molecules, recombinant viruses andrecombinant cells comprising such IL-18, caspase-1, and IL-12 singlechain nucleic acid molecules and methods to produce such nucleic acidmolecules, recombinant molecules, recombinant viruses and recombinantcells.

Another embodiment of the present invention is an isolated proteinselected from the group consisting of: (a) an isolated IL-18 proteinselected from the group consisting of: (i) an isolated protein of atleast 25 amino acids in length, wherein said protein has an at least 25contiguous amino acid region identical in sequence to a 25 contiguousamino acid region selected from the group consisting of SEQ ID NO:2, SEQID NO:5, SEQ ID NO:8 and SEQ ID NO:12; and (ii) an isolated proteinhaving an amino acid sequence that is at least 92 percent identical toan amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12 and a fragment thereofof at least 30 nucleotides; wherein said isolated protein has a functionselected from the group consisting of (i) eliciting an immune responseagainst an IL-18 protein having an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ IDNO:12, (ii) selectively binding to an antibody raised against an IL-18protein having an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12, and (iii)exhibiting IL-18 activity; (b) an isolated caspase-1 protein selectedfrom the group consisting of: (i) an isolated protein of at least about25 amino acids in length, wherein said protein has an at least 25contiguous amino acid region identical in sequence to a 25 contiguousamino acid region selected from the group consisting of SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24; and (ii) an isolatedprotein having an amino acid sequence that is at least 85 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24 and has anucleic acid fragment thereof of at least 30 nucleotides; wherein saidisolated protein has a function selected from the group consisting of(i) eliciting an immune response against a caspase-1 protein having anamino acid sequence selected from the group consisting of SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, (ii) ii) selectivelybinding to an antibody raised against caspase-1 protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, and SEQ ID NO:24, and (iii) exhibiting caspase-1activity; {circle around (C)}) an isolated IL-12 single chain proteincomprising an IL-12 p40 subunit domain linked to an IL-12 p35 subunitdomain, wherein said p40 subunit domain is selected from the groupconsisting of (i) a p40 subunit protein having an amino acid sequencethat is at least 84 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:27 and SEQ ID NO:30, (ii) a p40subunit protein comprising a fragment of a protein of (i), wherein saidfragment is at least 30 amino acids in length, and (iii) a p40 subunitprotein comprising at least 23 contiguous amino acids identical insequence to at least 23 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30 andwherein said p35 subunit domain is selected from the group consisting of(i) a p35 subunit protein having an amino acid sequence that is at least84 percent identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:33 and SEQ ID NO:36, (ii) a p35 subunit proteincomprising a fragment of a protein of (i), wherein said fragment is atleast 30 amino acids in length, and (iii) a p35 subunit proteincomprising at least 23 contiguous amino acids identical in sequence toat least 23 contiguous amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:33 and SEQ ID NO:36; wherein saidisolated protein has a function selected from the group consisting of(i) eliciting an immune response against an IL-12 protein having anamino acid sequence selected from the group consisting of SEQ ID NO:39and SEQ ID NO:44, (ii) selectively binding to an antibody raised againstan IL-12 protein having an amino acid sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36,SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQ IDNO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12 activity; and (d)an isolated IL-12 single chain protein comprising an IL-12 p40 subunitdomain linked to an IL-12 p35 subunit domain, wherein said p40 subunitdomain is selected from the group consisting of (i) a p40 subunitprotein having an amino acid sequence that is at least 84 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:53 and SEQ ID NO:59, (ii) a p40 subunit protein comprisinga fragment of a protein of (i), wherein said fragment is at least 40amino acids in length, and (iii) a p40 subunit protein comprising atleast 31 contiguous amino acids identical in sequence to at least 31contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:53 and SEQ ID NO:59; wherein said p35 subunitdomain is selected from the group consisting of (i) a p35 subunitprotein having an amino acid sequence that is at least 84 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35 subunit protein comprisinga fragment of a protein of (i), wherein said fragment is at least 40amino acids in length, and (iii) a p35 subunit protein comprising atleast 31 contiguous amino acids identical in sequence to at least 31contiguous amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:47 and SEQ ID NO:50; and wherein said isolatedprotein has a function selected from the group consisting of (i)eliciting an immune response against an IL-12 protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:39, SEQ IDNO:44, SEQ ID NO:62, and SEQ ID NO:67, (ii) selectively binding to anantibody raised against an IL-12 protein having an amino acid sequenceselected from the group consisting of SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQID NO:40, SEQ ID NO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12activity. The present invention also includes recombinant molecules,recombinant viruses and recombinant cells comprising such IL-18,caspase-1, and IL-12 single chain nucleic acid molecules and methods toproduce such nucleic acid molecules, recombinant molecules, recombinantviruses and recombinant cells.

The present invention also includes an antibody that selectively bindsto a protein of the present invention as well as methods to produce anduse such proteins or antibodies. By selectively is meant an antibodythat binds to a protein of the present invention, but does not bind asimilar protein of another species.

One aspect of the present invention is a therapeutic composition that,when administered to an animal, regulates an immune response in theanimal. Such a therapeutic composition includes at least one of thefollowing protective compounds: an IL-18, caspase-1, or IL-12 singlechain protein of the present invention, a mimetope of any of theproteins, a multimeric form of any of the proteins, an isolated nucleicacid molecule of the present invention, an antibody that selectivelybinds any of the proteins, and/or an inhibitor of a protein activityidentified by its ability to inhibit the activity of any of theproteins. Also included is a method to regulate an immune response byadministering such a therapeutic composition to an animal.

The present invention also includes a method to produce a protein of thepresent invention; such a method includes the step of culturing arecombinant cell capable of expressing a protein being encoded by anucleic acid molecule of the present invention.

Another embodiment of the present invention is a method to identify acompound capable of regulating an immune response in an animal, a methodselected from the group consisting of: (a) contacting an isolated felineIL-18 protein with a putative inhibitory compound under conditions inwhich, in the absence of the compound, the protein has T cellstimulating activity inducing T cells to make interferon gamma, anddetermining if the putative inhibitory compound inhibits that activity;(b) contacting an isolated feline caspase-1 protein with a putativeinhibitory compound under conditions in which, in the absence of thecompound, the protein cleaves precursor form of IL-18 resulting inbiologically active IL-18, and determining if the putative inhibitorycompound inhibits that activity; and {circle around (C)})) contacting anisolated IL-12 single chain protein with a putative inhibitory compoundunder conditions in which, in the absence of the compound, the proteinhas T cell proliferation stimulating activity, and determining if theputative inhibitory compound inhibits that activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated feline and canine proteins,nucleic acid molecules encoding such proteins, antibodies raised againstsuch proteins and/or inhibitors of such proteins or nucleic acidmolecules. Specifically, the present invention provides for isolatedfeline IL-18, feline caspase-1, and feline and canine IL-12 single chainproteins and nucleic acid molecules as well as antibodies raised againstsuch proteins, and/or inhibitors of such proteins or nucleic acidmolecules. Also included in the present invention is the use of theseproteins, nucleic acid molecules, antibodies, inhibitors and/or othercompounds derived therefrom as therapeutic compositions to regulate theimmune response of an animal as well as in other applications, such asthose disclosed below.

One embodiment of the present invention is an isolated protein thatincludes a feline IL-18 protein, a feline caspase-1 protein, a felineIL-12 single chain protein and/or a canine IL-12 single chain protein.As used herein, a feline and/or canine protein refers to a protein. Asused herein, a protein of the present invention is a protein that isisolated from a felid or a canid or is derived therefrom and can beproduced by methods known in the art, such as, for example, usingrecombinant DNA technology or by chemical synthesis. As such, a felineor canine protein of the present invention includes natural forms aswell as any variants thereof, such as a feline or canine protein thathas been altered in a manner known to those skilled in the art, such asthose methods disclosed herein. As used herein, a feline or canineprotein does not refer to a mouse or human protein.

Similarly, a feline or canine nucleic acid molecule of the presentinvention includes a feline IL-18 nucleic acid molecule, a felinecaspase-1 nucleic acid molecule, a feline IL-12 single chain nucleicacid molecule and/or canine IL-12 single chain nucleic acid molecule. Asused herein a feline or canine nucleic acid molecule of the presentinvention refers to a nucleic acid molecule that includes a nucleic acidmolecule that encodes a protein of the present invention and/or acomplement thereof. As such, a feline IL-18 nucleic acid molecule, afeline caspase-1 nucleic acid molecule, a feline IL-12 single chainnucleic acid molecule or a canine IL-12 single chain nucleic acidmolecule of the present invention is a nucleic acid molecule thatencodes a feline IL-18 protein, a feline caspase-1 protein, a felineIL-12 single chain protein or a canine IL-12 single chain protein,respectively, and/or that is a complement thereof. As used herein, afeline or canine nucleic acid molecule of the present invention is anucleic acid molecule that is isolated from a felid or canid or isderived therefrom and can be produced using methods known in the art,such as, for example, recombinant DNA technology, or by chemicalsynthesis. As such, a feline or canine nucleic acid molecule of thepresent invention includes natural forms as well as any variantsthereof, such as a feline or canine nucleic acid molecule that has beenaltered in a manner known to those skilled in the art, such as thosemethods disclosed herein. As used herein, a feline or canine nucleicacid molecule does not refer to a mouse or human nucleic acid molecule.

According to the present invention, an isolated, or biologically pure,nucleic acid molecule or protein, is a nucleic acid molecule or proteinthat has been removed from its natural milieu. As such, “isolated”and/or “biologically pure” do not necessarily reflect the extent towhich the nucleic acid molecule or protein has been purified. “Proteins”are defined as any compounds which comprise amino acids, includingpeptides, polypeptides and fusion proteins. It is to be noted that theterm “a” or “an” entity refers to one or more of that entity; forexample, a protein refers to one or more proteins or at least oneprotein. As such, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, and “having” can be usedinterchangeably. Furthermore, an item “selected from the groupconsisting of” refers to one or more of the items in that group,including combinations thereof. The term “fragment” refers to any subsetof the referent nucleic acid molecule. Furthermore, the term “linked inframe” refers to nucleic acid fragment joined to another nucleic acidfragment in a manner such that the molecule is able to be expressed whentransformed into a host cell.

As used herein, a felid refers to any member of the felid family (i.e.the family Felidae), including, but not limited to, domestic cats, andwild cats such as tigers, lions, and lynx. Similarly, the term felinerefers to “of the family Felidae”.

As used herein, a canid refers to any member of the canid family (i.e.the family Canidae), including, but not limited to, domestic dogs, andwild canids such as wolves, foxes, and coyotes. Similarly, the termcanine refers to “of the family Canidae”.

Nucleic acid molecules of the present invention of known length isolatedfrom Felis catus are denoted as follows: a feline IL-18 nucleic acidmolecule is denoted as nFeIL-18_(x), wherein “x” refers to the number ofnucleotides in that molecule; for example, nFeIL-18₆₀₇ refers to afeline IL-18 nucleic acid molecule of 607 nucleotides in length; and ina similar fashion, a feline Casp-1 nucleic acid molecule of length “x”is referred to as nFeCasp-1_(x), a feline IL-12 single chain nucleicacid molecule of length “x” is referred to as nFeIL-12_(x), a felineIL-12p35 subunit nucleic acid molecule of length “x” is referred to asnFeIL-12p35_(x) and a feline IL-12p40 subunit nucleic acid molecule oflength “x” is referred to as nFeIL-12p40_(x). Similarly, Felis catusIL-18, Casp-1, IL-12 single chain, IL-12p35 subunit, and IL-12p40subunit proteins of the present invention of known length are denotedPFeIL-18_(x), PFeCasp-1_(x), PFeIL-12_(x), PFeIL-12p35_(x), andPFeIL-12p40_(x) respectively.

Nucleic acid molecules of the present invention of known length isolatedfrom Canis familiaris are denoted as follows: a canine IL-12 singlechain is denoted as nCaIL-12_(x), wherein “x” refers to the number ofnucleotides in that molecule; for example, nCaIL-12₁₆₀₂ refers to acanine IL-12 single chain nucleic acid molecule of 1602 nucleotides inlength and in a similar fashion, a canine IL-12 single chain nucleicacid molecule of length “x” is referred to as nCaIL-12p35_(x) and acanine IL-12p40 subunit nucleic acid molecule of length “x” is referredto as nCaIL-12p40_(x). Similarly, Canis familiaris IL-12 single chainproteins of the present invention of known length isolated from aredenoted PCaIL-12_(x), PCaIL-12p35_(x), or PCaIL-12p40_(x) respectively.The present invention includes nucleic acid molecules selected from thegroup consisting of nCaIL-12p35₅₉₁, nCaIL-12p40₂₂₆₇, nCaIL-12p40₁₀₀₂,nCaIL-12p40₉₈₇, nCaIL-12₁₅₉₉, and nCaIL-12₁₅₃₃.

The present invention includes nucleic acid molecules that include oneor more of the following nucleic acid sequences: SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ IDNO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:61, SEQ IDNO:64 and/or SEQ ID NO:66, and/or a complements of these nucleic acidsequences, i.e. SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28,SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:45,SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:63,and/or SEQ ID NO:68, respectively. Complements are defined as two singlestrands of nucleic acid in which the nucleotide sequences are such thatthe strands will hybridize as a result of base pairing throughout theirfull length; i.e., these sequences are fully complementary. Such nucleicacid sequences are further described herein and can be easily bedetermined by those skilled in the art. It should be noted that sincenucleic acid sequencing technology is not entirely error-free, nucleicacid and protein sequences presented herein represent apparent nucleicacid and amino acid sequences of the isolated nucleic acid molecules andproteins, respectively, of the present invention.

As used herein, an isolated feline IL-18, feline caspase-1, felineIL-12p40 subunit, feline IL-12p35 subunit, feline IL-12 single chain,canine IL-12p40 subunit, canine IL-12p35, and/or canine IL-12 singlechain protein of the present invention can be a full-length protein orany homolog of such a protein, including truncated forms of the protein.An isolated IL-18 protein of the present invention, including a homolog,can be identified in a straight-forward manner by the protein's abilityto elicit an immune response against, (or to) an IL-18 protein, whetherthe protein has IL-18 activity, such as T cell stimulating activity, orselectively binding to an antibody raised against an IL-18 protein. Anisolated caspase-1 protein of the present invention may be identified ina straight-forward manner by the protein's ability to elicit an immuneresponse against, (or to) a caspase-1 protein, whether the protein hascaspase-1 activity, such as cleaving the precursor form of IL-18resulting in a biologically active IL-18, or selectively binding to anantibody raised against a caspase-1 protein. A IL-12 single chainprotein of the present invention may be identified in a straight-forwardmanner by the protein's ability to elicit an immune response against,(or to) an IL-12 protein, including the p35 or p40 subunits, whether theprotein has IL-12 activity, such as T cell stimulating activity, orselectively binding to an antibody raised against an IL-12 protein,including the p35 or p40 subunits. Examples of protein homologs of thepresent invention includes proteins of the present invention in whichamino acids have been deleted (e.g. a truncated version of the protein,such as a peptide), inserted, inverted, substituted and/or derivatized(e.g. by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol) such that the protein homolog include atleast one epitope capable of eliciting an immune response against theparent protein, where the term parent refers to the longer and/orfull-length protein that the homolog is derived from. The ability of aprotein to effect an immune response can be measured using techniquesknown to those skilled in the art. As used herein, the term “epitope”refers to the smallest portion of a protein capable of selectivelybinding to the antigen binding site of an antibody. It is well acceptedby those skilled in the art that the minimal size of a protein epitopecapable of selectively binding to the antigen binding site of anantibody is about five or six to seven amino acids.

Proteins of the present invention include variants of a full-lengthprotein of the present invention. Such variants include proteins thatare less than full-length. As used herein, variants of the presentinvention refer to nucleic acid molecules that are naturally-occurring adefined below, and may result from alternative RNA splicing, alternativetermination of an amino acid sequence or DNA recombination. Examples ofvariants include allelic variants as defined below. It is to be notedthat a variant is an example of a homolog of the present invention.

Proteins of the present invention are encoded by nucleic acid moleculesof the present invention. As used herein, an IL-18 nucleic acid moleculeincludes sequences related to a natural feline IL-18 gene. As usedherein, a caspase-1 protein includes nucleic acid sequences related to anatural feline caspase-1 gene. As used herein, an IL-12 single chainnucleic acid molecule includes sequences related to a natural canine orfeline IL-12 gene, IL-12p35 gene, and/or IL-12p40 gene. As used herein,a feline IL-18, a feline caspase-1, and or a feline or canine IL-12single chain refers to the natural genomic elements that encode a felineIL-18, a feline caspase-1, and or a feline or canine IL-12 single chain,and includes all regions such as regulatory regions that controlproduction of the protein encoded by the gene) such as, but not limitedto, transcription, translation or post-translation control regions) aswell as the coding region itself, and any introns or non-translatedcoding regions. As used herein, a gene that “includes” or “comprises” asequence may include that sequence in one continuous array, or mayinclude the sequence of fragmented exons. As used herein, the term“coding region” refers to a continuous linear array of nucleotides thattranslates into a protein. A full-length coding region is that regionthat is translated into a full-length, i.e., a complete protein as wouldbe initially translated in its natural milieu, prior to anypost-translational modifications.

In one embodiment of the present invention, isolated proteins areencoded by nucleic acid molecules that hybridize under stringenthybridization conditions to the non-coding strand of nucleic acidmolecules encoding proteins. The minimal size of a protein of thepresent invention (4-6 amino acids) is a size sufficient to be encodedby a nucleic acid molecule capable of forming a stable hybrid, i.e.,hybridizing under stringent hybridization conditions, with thecomplementary sequence of a nucleic acid molecule encoding thecorresponding natural protein. The size of a nucleic acid moleculeencoding such a protein is dependent on the nucleic acid composition andthe percent homology between the nucleic acid molecule and thecomplementary nucleic acid sequence. It can easily be understood thatthe extent of homology required to form a stable hybrid under stringentconditions can vary depending on whether the homologous sequences areinterspersed throughout a given nucleic acid molecule or are clustered,i.e. localized, in distinct regions on a given nucleic acid molecule.

The minimal size of a feline IL-18, feline caspase-1, and/or canine orfeline IL-12 single chain protein homolog/portion/fragment of thepresent invention is a size sufficient to be encoded by a nucleic acidmolecule capable of forming a stable hybrid (i.e. hybridize understringent hybridization conditions) with the complementary sequence of anucleic acid molecule encoding the corresponding natural protein.Stringent hybridization conditions are determined based on definedphysical properties of the gene to which the nucleic acid molecule isbeing hybridized, and can be defined mathematically. Stringenthybridization conditions are those experimental parameters that allow anindividual skilled in the art to identify significant similaritiesbetween heterologous nucleic acid molecules. These conditions are wellknown to those skilled in the art. See, for example, Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284, each ofwhich is incorporated herein by this reference. As explained in detailin the cited references, the determination of hybridization conditionsinvolves the manipulation of a set of variables including the ionicstrength (M, in moles/liter), the hybridization temperature (° C.), theconcentration of nucleic acid helix destabilizing agents, such asformamide, the average length of the shortest hybrid duplex (n), and thepercent G+C composition of the fragment to which an unknown nucleic acidmolecule is being hybridized. For nucleic acid molecules of at leastabout 150 nucleotides, these variables are inserted into a standardmathematical formula to calculate the melting temperature, or T_(m), ofa given nucleic acid molecule. As defined in the formula below, T_(m) isthe temperature at which two complementary nucleic acid molecule strandswill disassociate, assuming 100% complementarity between the twostrands:T _(m)=81.5° C.+16.6 log M+0.41(%G+C)−500/n−0.61(%formamide).For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:T _(d)=4(G+C)+2(A+T).A temperature of 5° C. below T_(d) is used to detect hybridizationbetween perfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch,i.e. differences between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base pairs forhybrids greater than about 150 bp, and T_(d) decreases about 5° C. foreach mismatched base pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions, by altering, for example, the salt concentration, theformamide concentration or the temperature, so that only nucleic acidhybrids with greater than a specified % base pair mismatch willhybridize. Stringent hybridization conditions are commonly understood bythose skilled in the art to be those experimental conditions that willallow about 30% or less base pair mismatch, i.e., at least about 85%identity. Because one skilled in the art can easily determine whether agiven nucleic acid molecule to be tested is less than or greater thanabout 50 nucleotides, and can therefore choose the appropriate formulafor determining hybridization conditions, he or she can determinewhether the nucleic acid molecule will hybridize with a given gene orspecified nucleic acid molecule under stringent hybridization conditionsand similarly whether the nucleic acid molecule will hybridize underconditions designed to allow a desired amount of base pair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

In one embodiment, an IL-18 gene of the present invention includes thenucleic acid molecule SEQ ID NO:1, as well as the complement of SEQ IDNO:1. Nucleic acid sequence SEQ ID NO:1 represents the deduced sequenceof the coding strand of a cDNA (complementary DNA) denoted herein asnucleic acid molecule nFeIL-18-N₅₁₄, the production of which isdisclosed in the Examples. SEQ ID NO:1 comprises an apparent partialcoding region of nFeIL-18, coding for the N-terminal portion of felineIL-18 protein. The complement of SEQ ID NO:1 (represented herein by SEQID NO:3) refers to the nucleic acid sequence of the strand fullycomplementary to the strand having SEQ ID NO:1, which can easily bedetermined by those skilled in the art. Likewise, a nucleic acidsequence complement of any nucleic acid sequence of the presentinvention refers to the nucleic acid sequence complement of any nucleicacid sequence of the present invention tat is fully complementary (i.e.can form a double helix with) the strand for which the sequence iscited. It should be noted that since nucleic acid sequencing technologyis not entirely error-free, SEQ ID NO:1 (as well as other nucleic acidand protein sequences presented herein) represents an apparent nucleicacid sequence of the nucleic acid molecule encoding an immunoregulatoryprotein of the present invention.

Another IL-18 gene of the present invention includes the nucleic acidmolecule SEQ ID NO:4, as well as the complement represented by SEQ IDNO:6. Nucleic acid sequence SEQ ID NO:4 represents the deduced sequenceof the coding strand of a cDNA denoted herein as nucleic acid moleculenFeIL-18-C₅₀₂, the production of which is disclosed in the examples.Nucleic acid nFeIL-18-C₅₀₂ represents an apparent partial coding regionof FeIL-18, encoding a partial C-terminal region of the feline IL-18protein. Another IL-18 gene of the present invention includes thenucleic acid molecule SEQ ID NO:7, as well as the complement representedby SEQ ID NO:10. Nucleic acid sequence SEQ ID NO:7 represents thededuced sequence of the coding strand of a cDNA denoted herein asnucleic acid molecule nFeIL-18₆₀₇, the production of which is disclosedin the examples. Nucleic acid nFeIL-18₆₀₇ represents an apparentfull-length coding region of the feline IL-18 protein. Another IL-18gene of the present invention includes the nucleic acid molecule SEQ IDNO:9, as well as the complement represented by SEQ ID NO:41. Nucleicacid sequence SEQ ID NO:9 represents the deduced sequence of the codingstrand of a cDNA denoted herein as nucleic acid molecule nFe IL-18₅₇₆,the production of which is disclosed in the examples. Nucleic acidmolecule nFe IL-18₅₇₆ represents the coding region for an apparentprecursor protein to a mature feline IL-18 protein. Another IL-18 geneof the present invention includes the nucleic acid molecule SEQ IDNO:11, as well as the complement represented by SEQ ID NO:13. Nucleicacid sequence SEQ ID NO:11 represents the deduced sequence of the codingstrand of a cDNA denoted herein as nucleic acid molecule nFe IL-18₄₇₁,the production of which is disclosed in the Examples. Nucleic acidmolecule nFe IL-18₄₇₁ represents the coding region for an apparentmature IL-18 protein. The putative cleavage site for the mature IL-18protein is between amino acid positions 35 and 36 of SEQ ID NO:8,representing PFeIL-18₁₉₂, which is the predicted amino acid sequence ofthe full-length IL-18 protein (i.e., containing signal, or leader,peptide). SEQ ID NO:12 represents the predicted amino acid sequence ofthe mature IL-18 protein (i.e., without the signal, or leader,sequence), also denoted as PFeIL-18₁₅₇.

In another embodiment, a caspase-1 gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:14, as well as thecomplement represented by SEQ ID NO:16. Nucleic acid sequence SEQ IDNO:14 represents the deduced sequence of the coding strand of a cDNAdenoted herein as nucleic acid molecule nFeCasp-1₁₂₃₃, the production ofwhich is disclosed in the Examples. Nucleic acid molecule nFeCasp-1₁₂₃₃represents the coding region for an apparent full-length felinecaspase-1 protein and includes a human primer sequence. Anothercaspase-1 protein of the present invention includes the nucleic acidsequence SEQ ID NO:17, as well as the complement represented by SEQ IDNO:19. Nucleic acid sequence SEQ ID NO:17 represents the deducedsequence of the coding strand of a cDNA denoted herein as nucleic acidmolecule nFeCasp-1-N₅₂₆, the production of which is disclosed in theExamples. Nucleic acid molecule nFeCasp-1-N₅₂₆ represents the codingregion for the apparent N-terminal region of the feline caspase-1protein. Another caspase-1 protein of the present invention includes thenucleic acid molecule SEQ ID NO:20, as well as the complementrepresented by SEQ ID NO:22. Nucleic acid sequence SEQ ID NO:20represents the deduced sequence of a coding strand of a cDNA denotedherein as nucleic acid molecule nFeCasp-1-C₅₀₀, the production of whichis disclosed in the Examples. Nucleic acid molecule nFeCasp-1-C₅₀₀represents the coding region for the apparent C-terminal region of thefeline caspase-1 protein. Another caspase-1 protein of the presentinvention includes the nucleic acid molecule SEQ ID NO:23, as well asthe complement represented by SEQ ID NO:25. Nucleic acid sequence SEQ IDNO:23 represents the deduced sequence of a coding strand of a cDNAdenoted herein as nucleic acid molecule nFeCasp-1₁₂₃₀, the production ofwhich is disclosed in the examples. Nucleic acid molecule nFeCasp-1₁₂₃₀represents the coding region for the apparent full-length felinecaspase-1 protein, denoted herein as PFeCasp-1₄₁₀, represented herein asSEQ ID NO:24.

In another embodiment, feline IL-12 single chain proteins of the presentinvention contain both a mature IL-12p35 subunit and a full-lengthIL-12p40 subunit, joined by a linker. An IL-12 single chain gene of thepresent invention includes the nucleic acid sequence SEQ ID NO:38, aswell as the complement represented by SEQ ID NO:40. Nucleic acidsequence SEQ ID NO:38 represents the deduced sequence of the codingstrand of a cDNA denoted herein as nucleic acid molecule nFeIL-12₁₅₉₉,the production of which is disclosed in the Examples. Nucleic acidmolecule nFeIL-12₁₅₉₉ represents the coding region encoding a singlechain full-length feline IL-12 protein, which includes the coding regionfor a full-length (i.e. containing signal, or leader, sequence) IL-12p40subunit, a linker of the present invention, and the coding region for amature (i.e. not containing signal, or leader, sequence) IL-12p35subunit. SEQ ID NO:38 comprises a sequence that includes both thenucleic acid sequence SEQ ID NO:29 (nucleic acid sequence SEQ ID NO:29represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeIL-12p40₉₈₇, which represents thecoding region encoding the full-length feline IL-12p40 subunit, whereasSEQ ID NO:31 represents the complement of SEQ ID NO:29) and SEQ ID NO:35(nucleic acid sequence SEQ ID NO:35 represents the deduced sequence ofthe coding strand of a cDNA denoted herein as nucleic acid moleculenFeIL-12p35₅₉₁, which represents the coding region encoding the maturefeline IL-12p35 subunit, whereas SEQ ID NO:37 represents the complementof SEQ ID NO:35). Translation of SEQ ID NO:38 yields a predicted proteindenoted herein as PFeIL-12₅₃₃, also denoted as SEQ ID NO:39.

In another embodiment, feline IL-12 single chain proteins of the presentinvention contain both a mature IL-12p35 subunit and an mature IL-12p40subunit, joined by a linker. An IL-12 single chain gene of the presentinvention includes the nucleic acid sequence SEQ ID NO:43, as well asthe complement represented by SEQ ID NO:45. Nucleic acid sequence SEQ IDNO:43 represents the deduced sequence of the coding strand of a cDNAdenoted herein as nucleic acid molecule nFeIL-12₁₅₃₃, the production ofwhich is disclosed in the Examples. Nucleic acid molecule nFeIL-12₁₅₃₃represents the coding region encoding a single chain mature feline IL-12protein. SEQ ID NO:33 comprises a sequence that includes both thenucleic acid sequence SEQ ID NO:26 (nucleic acid sequence SEQ ID NO:26represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeIL-12p40₉₈₅, which represents thecoding region encoding the mature feline IL-12p40 subunit, whereas SEQID NO:28 represents the complement of SEQ ID NO:26) and SEQ ID NO:35(nucleic acid sequence SEQ ID NO:35 represents the deduced sequence ofthe coding strand of a cDNA denoted herein as nucleic acid moleculenFeIL-12p35₅₉₁, which represents the coding region encoding the maturefeline IL-12p35 subunit, whereas SEQ ID NO:37 represents the complementof SEQ ID NO:35). Translation of SEQ ID NO:43 yields a predicted proteindenoted herein as PFeIL-12₅₁₁, also denoted as SEQ ID NO:44.

In another embodiment, canine IL-12 single chain proteins and nucleicacid molecules of the present invention contain both a mature IL-12p35subunit and a full-length IL-12p40 subunit, joined by a linker. An IL-12single chain gene of the present invention includes the nucleic acidsequence SEQ ID NO:61, as well as the complement represented by SEQ IDNO:63. Nucleic acid sequence SEQ ID NO:61 represents the deducedsequence of the coding strand of a cDNA denoted herein as nucleic acidmolecule nCaIL-12₁₅₉₉, the production of which is disclosed in theExamples. Nucleic acid molecule nCaIL-12₁₅₉₉ represents the codingregion encoding a single chain full-length canine IL-12 protein, whichincludes the coding region for a full-length (i.e. containing signal, orleader, sequence), IL-14p40 subunit, a linker of the present invention,and the coding region for a mature, (i.e., not containing signal, orleader, sequence) IL-12p35 subunit. SEQ ID NO:61 comprises a nucleicacid sequence that includes both the nucleic acid sequence SEQ ID NO:58(nucleic acid SEQ ID NO:58 represents the deduced sequence of the codingstrand of a cDNA denoted herein as nucleic acid molecule nCaIL-12₉₈₇,which represents the coding region encoding the full-length canineIL-12p40 subunit, whereas SEQ ID NO:60 represents the complement of SEQID NO:58) and SEQ ID NO:49 (nucleic acid sequence SEQ ID NO:49represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeIL-12p35₅₉₁, which represents thecoding region encoding the mature canine IL-12 subunit, whereas SEQ IDNO:51 represents the complement of SEQ ID NO:49. Translation of SEQ IDNO:61 yields a predicted protein denoted herein as PCaIL-12₅₃₃, alsodenoted as SEQ ID NO:62.

In another embodiment, canine IL-12 single chain proteins and nucleicacid molecules of the present invention contain both a mature IL-12p35subunit and a mature IL-12p40 subunit, joined by a linker. An IL-12single chain gene of the present invention includes the nucleic acidsequence SEQ ID NO:66, as well as the complement represented by SEQ IDNO:68. Nucleic acid sequence SEQ ID NO:66 represents the deducedsequence of the coding strand of a cDNA denoted herein as nucleic acidmolecule nCaIL-12,₁₅₃₃, the production of which is disclosed in theExamples. Nucleic acid molecule nCaIL-12₁₅₃₃ represents the codingregion encoding a single chain full-length canine IL-12 protein, whichincludes the coding region for a full-length (i.e. containing signal, orleader, sequence), IL-14p40 subunit, a linker of the present invention,and the coding region for a mature, (i.e., not containing signal, orleader, sequence) IL-12p35 subunit. SEQ ID NO:66 comprises a nucleicacid sequence that includes both the nucleic acid sequence SEQ ID NO:52(nucleic acid SEQ ID NO:52 represents the deduced sequence of the codingstrand of a cDNA denoted herein as nucleic acid molecule nCaIL-12₉₂₁,which represents the coding region encoding the mature canine IL-12p40subunit, whereas SEQ ID NO:54 represents the complement of SEQ ID NO:52)and SEQ ID NO:49 (nucleic acid sequence SEQ ID NO:49 represents thededuced sequence of the coding strand of a cDNA denoted herein asnucleic acid molecule nFeIL-12p35₅₉₁, which represents the coding regionencoding the mature canine IL-12 subunit, whereas SEQ ID NO:51represents the complement of SEQ ID NO:49. Translation of SEQ ID NO:66yields a predicted protein denoted herein as PCaIL-12₅₁₁, also denotedas SEQ ID NO:67.

Nucleic acid molecules and proteins of the present invention havingspecific sequence identifiers are described in Table 1. TABLE 1 Sequenceidentification numbers (SEQ ID NOs) and their corresponding nucleic acidmolecule or proteins. SEQ ID NO: description 1 nFeIL-18-N₅₁₄ codingstrand 2 PFeIL-18-N₁₃₃ 3 nFeIL-18-N₅₁₄ complementary strand 4nFeIL-18-C₅₀₂ coding strand 5 PFeIL-18-C₁₅₄ 6 nFeIL-18-C₅₀₂complementary strand 7 nFeIL-18₆₀₇ coding strand 8 PFeIL-18₁₉₂ 9nFeIL-18₅₇₆ coding strand: coding sequence for full-length feline IL-18protein 10 nFeIL-18₆₀₇ complementary strand to SEQ ID NO: 7 11nFeIL-18₄₇₁ coding strand: coding sequence for mature feline IL-18protein 12 PFeIL-18₁₅₇ 13 nFeIL-18₄₇₁ complementary strand 14nFeCasp-1₁₂₃₃ coding strand 15 PFeCasp-1₄₁₀ 16 nFeCasp-1₁₂₃₃complementary strand 17 nFeCasp-1-N₅₂₆ coding strand 18 PFeCasp-1-N₁₆₉19 nFeCasp-1-N₅₂₆ complementary strand 20 nFeCasp-1-C₅₀₀ coding strand21 PFeCasp-1-C₁₂₀ 22 nFeCasp-1-C₅₀₀ complementary strand 23nFeCasp-1₁₂₃₀ coding strand: coding sequence for feline caspase-1protein 24 PFeCasp-1₄₁₀ 25 nFeCasp-1₁₂₃₀ complementary strand 26nFeIL-12p40₉₂₁ coding strand: coding sequence for feline mature IL-12p40subunit 27 PFeIL-12p40₃₀₇ 28 nFeIL-12p40₉₂₁ complementary strand 29nFeIL-12p40₉₈₇ coding strand: coding sequence for feline full-lengthIL-12p40 subunit 30 PFeIL-12p40₃₂₉ 31 nFeIL-12p40₉₈₇ complementarystrand 32 nFeIL-12p35₆₆₆ coding strand: coding sequence for felinefull-length IL-12p35 subunit 33 PFeIL-12p35₂₂₂ 34 nFeIL-12p35₆₆₆complementary strand 35 nFeIL-12p35₅₉₁ coding strand: coding sequencefor feline mature IL-12p35 subunit 36 PFeIL-12p35-N₁₈₇ 37 nFeIL-12p35₅₉₁complementary strand 38 nFeIL-12₁₅₉₉ coding strand 39 PFeIL-12₅₃₃ 40nFeIL-12₁₅₉₉ complementary strand 41 nFeIL-18₅₇₆ complementary strand toSEQ ID NO: 9 42 not used-inactive 43 nFeIL-12₁₅₃₃ coding strand 44PFeIL-12₅₁₁ 45 nFeIL-12₁₅₃₃ complementary strand 46 nCaIL-12p35₆₆₆coding strand: coding strand for canine full-length IL-12p35 subunit 47PCaIL-12p35₂₂₂ 48 nCaIL-12p35₆₆₆ complementary strand 49 nCaIL-12p35₅₉₁coding strand 50 PCaIL-12p35₁₉₇ 51 nCaIL-12p35₅₉₁ complementary strand52 nCaIL-12p40₉₂₁ coding strand: coding sequence for mature form canineIL-12 p40 subunit 53 PCaIL-12p40₃₀₇ 54 nCaIL-12p40₉₂₁ reverse complement55 nFeIL-12p40-N₉₈₅ coding sequence 56 PCaIL-12p40-N₃₂₈ 57nFeIL-12p40-N₉₈₅ complementary strand 58 nCaIL-12p40₉₈₇ coding strand:coding sequence for full-length canine IL-12 p40 subunit 59PCaIL-12p40₃₂₉ 60 nCaIL-12p40₉₈₇ complementary strand 61 nCaIL-12₁₅₉₉coding strand 62 PCaIL-12₅₃₃ 63 nCaIL-12₁₅₉₉ complementary strand 64 notused-inactive 65 not used-inactive 66 nCaIL-12₁₅₃₃ coding strand 67PCaIL-12₅₁₁ 68 nCaIL-12₁₅₃₃ complementary strand 101 nFeIL-12p35-N₅₆₁coding strand 102 PFeIL-12p35-N₁₈₇ 103 nFeIL-12p35-N₅₆₁ complementarystrand 104 nCaIL-12p35₁₄₅₅ coding strand 105 PCaIL-12p35₂₂₂ 106nCaIL-12p35₁₄₅₅ complementary strand 107 nCaIL-12p40₂₂₆₇ coding strand108 PcaIL-12p40₃₂₉ 109 nCaIL-12p40₂₂₆₇ complementary strand

Particularly preferred nucleic acid molecules encoding feline IL-18proteins are nFeIL-18-N₅₁₄, nFeIL-18-C₅₀₂, nFeIL-18₆₀₇, nFeIL-18₅₇₆, andnFeIL-18₄₇₁, the coding strands of which are represented by SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, respectively.

Particularly preferred nucleic acid molecules encoding feline caspase-1proteins are nFeCasp-1₁₂₃₃, nFeCasp-1-N₅₂₆, nFeCasp-1-C₅₀₀ andnFeCasp-1₁₂₃₀, the coding strands of which are represented by SEQ IDNO:14, SEQ ID NO:17, SEQ ID NO:20, and SEQ ID NO:23 respectively.

Particularly preferred nucleic acid molecules encoding canine and felineIL-12p35 and p40 subunit proteins are nFeIL-12p40-N₉₈₅, nFeIL-12p40₉₈₇,nFeIL-12p40₉₂₁, nFeIL-12p35₆₆₆, nFeIL-12p35-N₅₆₁, nFeIL-12p35₅₉₁,nCaIL-12p35₆₆₆, nCaIL-12p35₁₄₅₅, nCaIL-12p35₅₉₁, nCaIL-12p40₂₂₆₇,nCaIL-12p40₉₂₁, and nCaIL-12p40₉₈₇. Coding strands of which arerepresented by SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35,SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58,SEQ ID NO:101, SEQ ID NO:104, and SEQ ID NO:107.

Additional preferred nucleic acid molecules encoding canine and felineIL-12 single chain proteins are nFeIL-12₁₅₃₃, nFeIL-12₁₅₉₉,nCaIL-12₁₅₃₃, nCaIL-12₁₅₉₉, the coding strands of which are representedby SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:61, and SEQ ID NO:66.

One embodiment of the present invention includes an isolated nucleicacid molecule that is selected from a group of nucleic acid molecules.One member of this group includes an isolated nucleic acid molecule thatis selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ IDNO:41, SEQ ID NO:11, and SEQ ID NO:13; and a nucleic acid moleculecomprising at least 70 contiguous nucleotides identical in sequence toat least 70 contiguous nucleotides of a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ IDNO:11, and SEQ ID NO:13. Another member of this group of nucleic acidmolecules includes an isolated nucleic acid molecule that is selectedfrom the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, and SEQ IDNO:25; and a nucleic acid molecule comprising at least 70 contiguousnucleotides identical in sequence to at least 70 contiguous nucleotidesof a nucleic acid sequence selected from the group consisting of SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:23, and SEQ ID NO:25. Another member of this group ofnucleic acid molecules includes an isolated nucleic acid molecule thatis selected from the group consisting of a nucleic acid moleculecomprising (a) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:26, SEQ IDNO:29, and a nucleic acid sequence comprising at least 44 contiguousnucleotides identical in sequence to at least 44 contiguous nucleotidesof a nucleic acid sequence selected from the group consisting of SEQ IDNO:26 and SEQ ID NO:9; (b) a nucleic acid linker of (XXX)_(n) whereinn=0 to 60; and (c) an isolated nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ IDNO:32, SEQ ID NO:35, and a nucleic acid molecule comprising at least 44contiguous nucleotides identical in sequence to at least 44 contiguousnucleotides of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:32 and SEQ ID NO:35, such that a nucleic acidmolecule of this particular group encodes a feline IL-12protein. Anothermember of this group of nucleic acid molecules includes an isolatenucleic acid molecule selected from the group consisting of a nucleicacid molecule comprising (a) an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:52 and SEQ ID NO:58, and a nucleic acid sequence comprising atleast 47 contiguous nucleotides identical in sequence to at least 47contiguous nucleotides of a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:46 and SEQ ID NO:49; (b) a nucleic acidlinker of (XXX)_(n) wherein n=0 to 60; and {circle around (C)}) anisolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:46, SEQ ID NO:49, and anucleic acid molecule comprising at least 47 contiguous nucleotidesidentical in sequence to at least 47 contiguous nucleotides of a nucleicacid sequence selected from the group consisting of SEQ ID NO:46 and SEQID NO:49, such that a nucleic acid molecule of this particular groupencodes a canine IL-12 single chain protein.

The phrase, a nucleic acid molecule comprising at least “x” contiguousnucleotides identical in sequence to at least “x” contiguous nucleotidesof a nucleic acid molecule selected from the group consisting of SEQ IDNO:“y”, refers to an “x”-nucleotide in length nucleic acid molecule thatis identical in sequence to an “x”-nucleotide portion of SEQ ID NO:“y”,as well as to nucleic acid molecules that are longer in length than “x”.The additional length may be in the form of nucleotides that extend fromeither the 5′ or the 3′ end(s) of the contiguous identical“x”-nucleotide portion. The 5′ and/or 3′ extensions can include one ormore extensions that have no identity to an immunoregulatory molecule ofthe present invention, as well as extensions that show similarity oridentity to cited nucleic acids sequences or portions thereof.

Preferred portions, or lengths, for feline IL-18, feline caspase-1,feline IL-12 single chain, and canine IL-12 single chain nucleic acidmolecules of the present invention include nucleic acid molecules of atleast 40 nucleotides in length, at least 43 nucleotides in length, atleast 44 nucleotides in length, at least 47 nucleotides in length, atleast 50 nucleotides in length, at least 55 nucleotides in length, atleast 60 nucleotides in length, at least 65 nucleotides in length, atleast 70 nucleotides in length, at least 75 nucleotides in length, atleast 80 nucleotides in length, at least 85 nucleotides in length, atleast 90 nucleotides in length, at least 95 nucleotides in length, atleast 100 nucleotides in length, at least 120 nucleotides in length, atleast 140 nucleotides in length, at least 160 nucleotides in length, atleast 180 nucleotides in length, at least 200 nucleotides in length, atleast 250 nucleotides in length, at least 300 nucleotides in length, atleast 350 nucleotides in length, at least 400 nucleotides in length, atleast 450 nucleotides in length, at least 500 nucleotides in length, atleast 600 nucleotides in length, at least 700 nucleotides in length, atleast 800 nucleotides in length, at least 900 nucleotides in length, anda full-length molecule. Particularly preferred portions, or lengths, ofthe nucleic acid molecules of the present invention include nucleicacids of at least 43 nucleotides, 44 nucleotides, 47 nucleotides, 70nucleotides, and a full length molecule.

One embodiment of a protein and/or nucleic acid molecule of the presentinvention is a fusion nucleic acid and/or protein that includes either afeline IL-18, caspase-1, feline IL-12 single chain, and canine IL-12single chain nucleic acid molecule and/or protein of the presentinvention domain, each attached to one or more fusion segments. Suitablefusion segments for use with the present invention include, but are notlimited to, segments that can: link two or more nucleic acids and/orproteins of the present invention, to form multimeric forms of a nucleicacids and/or protein of the present invention; enhance a nucleic acidmolecules or protein's stability; enhance the biological activity of anucleic acid molecule and/or protein of the present invention; and/orassist in purification a molecule of the present invention (e.g., byaffinity chromatography). A suitable fusion segment can be a domain ofany size that has the desired function (e.g., imparts increasedstability, enhanced activity, and/or simplifies purification of aprotein). Fusion segments can be joined to amino and/or carboxyl terminiof the IL-18-containing domain, or the caspase-1 ligand-containingdomain, or the IL-12p40-containing domain, or the IL-12p35-containingdomain, or the IL-12 single chain-containing domain, of a protein and/ornucleic acid and can be susceptible to cleavage in order to enablestraight-forward recovery of the protein and/or nucleic acid molecule.Fusion proteins are preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid molecule that encodes a proteinincluding the fusion segment attached to either the carboxyl and/oramino terminal end of a feline IL-18, feline caspase-1, feline IL-12p35subunit, feline IL-12p40 subunit, feline IL-12 single chain, canineIL-12p35 subunit, canine IL-12p40 subunit, and/or canine IL-12 singlechain-containing domain. Preferred fusion segments include a metalbinding domain (e.g., a poly-histidine segment); an immunoglobulinbinding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptoror complement protein antibody-binding domains); a sugar binding domain(e.g., a maltose binding domain); and/or a “tag” domain (e.g., at leasta portion of β-galactosidase, a strep tag peptide, a T7 tag peptide, aFlag™ peptide, or other domains that can be purified using compoundsthat bind to the domain, such as monoclonal antibodies). More preferredfusion segments include metal binding domains, such as a poly-histidinesegment; a maltose binding domain; a strep tag peptide, such as thatavailable from Biometra in Tampa, Fla.; and an S10 peptide.

The phrase, a nucleic acid linker, is a term known to those skilled inthe art, and refers to a nucleic acid linker that can link, or attach,nucleic acid molecules, in such a manner that expression of the nucleicacid molecules produces one fusion protein as expression product. Alinker can be any nucleotide sequence that directs expression of asingle fusion polypeptide from a nucleotide molecule which includes twoor more nucleic acid molecules of the present invention, wherein thefusion polypeptide has appropriate biological activity. Preferably, anucleic acid linker of the present invention comprises nucleotidesarranged in codons, (i.e., 3 nucleotides that, when transcribed, codefor an amino acid residue), and the linker does not contain any stopcodons in frame. A linker is represented herein as (XXX)_(n), where X isthe designation of a variable nucleotide and n refers to the number ofcodons. The length of the nucleic acid linker may be of any length thatpermits expression of the fusion protein. More preferably, the length ofthe nucleic acid linker is from about 0 codons to about 60 codons, orfrom about 0 nucleotides to about 180 nucleotides. A particularlypreferred linker includes SEQ ID NO:83. Appropriate biological activityincludes the ability of such a fusion protein to elicit an immuneresponse against a protein of the present invention, selectively bindingan antibody raised against a protein of the present invention, andexhibiting the immunoregulatory activity of a protein of the presentinvention.

A single chain IL-12 protein of the present invention includes singlechain IL-12 proteins comprising an IL-12p35 subunit of the presentinvention at the N-terminus of the single chain protein and an IL-12p40subunit of the present invention at the C-terminus of the single chainprotein, with the linker between the p35 subunit and the p40 subunit.Preferred single chain IL-12 proteins comprise an IL-12p40 of thepresent invention at the N-terminus of the single chain protein and anIL-12p40 subunit of the present invention at the C-terminus of thesingle chain protein, with the linker in between the subunits.

Another embodiment of the present invention includes an isolated nucleicacid molecule that is selected from the group consisting of: (i) anucleic acid molecule having a nucleic acid sequence that is at least 92percent identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ ID NO:11, and SEQ IDNO:13; and (ii) a nucleic acid molecule comprising a fragment of anucleic acid molecule of (i) wherein said fragment is at least 80nucleotides in length. Preferred nucleic acid molecules include nucleicacid sequences that are at least 92%, at least 93%, at least 94%, morepreferably at least 95% identical, and even more preferably at leastabout 98% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ IDNO:11, and SEQ ID NO:13. Preferred fragment lengths include fragments ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ ID NO:1, and SEQ ID NO:13 whichare at least 75 nucleotides in length, which are at least 80 nucleotidesin length, which are at least 85 nucleotides in length, which are atleast 90 nucleotides in length, which are at least 100 nucleotides inlength, which are at least 120 nucleotides in length, which are at least150 nucleotides in length, which are at least 200 nucleotides in length,which are at least 300 nucleotides in length, which are at least 400nucleotides in length, which are at least 500 nucleotides in length,which are at least 600 nucleotides in length, and which preferably arefull-length.

Another embodiment of the present invention includes an isolated nucleicacid molecule that is selected from the group consisting of: (i) anucleic acid molecule having a nucleic acid sequence that is at least 85percent identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:25; and (ii) anucleic acid molecule comprising a fragment of a nucleic acid moleculeof (i) wherein said fragment is at least 85 nucleotides in length.Preferred nucleic acid molecules include nucleic acid sequences that areat least 85%, preferably at least 87%, more preferably at least 90%,even more preferably at least 95% identical to SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:25. Preferred fragment lengths include fragments ofSEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:25 which are at least 70nucleotides in length, which are at least 80 nucleotides in length,which are at least 85 nucleotides in length, which are at least 90nucleotides in length, which are at least 100 nucleotides in length,which are at least 200 nucleotides in length, which are at least 300nucleotides in length, which are at least 400 nucleotides in length,which are at least 500 nucleotides in length, which are at least 600nucleotides in length, or which preferably are full-length.

Another embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (i) a nucleic acidmolecule comprising (a) a nucleic acid molecule comprising a nucleicacid sequence that is at least 87 percent identical to a nucleic acidsequence selected from the group consisting of SEQ ID NO:26 and SEQ IDNO:29, or a fragment thereof of at least 55 nucleotides in length; (b) anucleic acid linker of (XXX)_(n) wherein n=0 to 60, and {circle around(C)}) a nucleic acid molecule comprising a nucleic acid sequence that isat least 87 percent identical to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:32 and SEQ ID NO:35, or a fragmentthereof of at least 55 nucleotides in length, such that said nucleicacid molecule of (i) encodes a feline IL-12 single chain protein; and anucleic acid molecule fully complementary to the coding strand of anucleic acid molecule as set forth in (i). Preferred nucleic acidmolecules include nucleic acid sequences that are at least 87%, at least88%, at least 89%, more preferably at least 90%, even more preferably atleast 95% identical to SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, and SEQID NO:35. Preferred fragment lengths include fragments of SEQ ID NO:26,SEQ ID NO:29, SEQ ID NO:32, and SEQ ID NO:35, which are at least 55nucleotides in length, which are at least 60 nucleotides in length,which are at least about 65 nucleotides in length, which are at least 70nucleotides in length, which are at least 80 nucleotides in length,which are at least 90 nucleotides in length, which are at least 100nucleotides in length, which are at least 200 nucleotides in length,which are at least 300 nucleotides in length, which are at least 400nucleotides in length, which are at least 500 nucleotides in length,which are at least 600 nucleotides in length, or which preferably arefull-length.

Another embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (i) a nucleic acidmolecule comprising (a) a nucleic acid molecule comprising a nucleicacid sequence that is at least 87 percent identical to a nucleic acidsequence selected from the group consisting of SEQ ID NO:52 and SEQ IDNO:58, or a fragment thereof of at least 55 nucleotides in length; (b) anucleic acid linker of (XXX)_(n) wherein n=0 to 60; and {circle around(C)}) a nucleic acid molecule comprising a nucleic acid sequence that isat least 87 percent identical to a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:46 and SEQ ID NO:49, or a fragmentthereof of at least 55 nucleotides in length, such that said nucleicacid molecule of (i) encodes a canine IL-12 single chain protein; and anucleic acid molecule fully complementary to the coding strand of anucleic acid molecule as set forth in (i). Preferred nucleic acidmolecules include nucleic acid sequences that are at least 87%, at least88%, at least 89%, more preferably at least 90%, even more preferably atleast 95% identical to SEQ ID NO:52, SEQ ID NO:58, SEQ ID NO:46, and SEQID NO:49. Preferred fragment lengths include fragments of SEQ ID NO:52,SEQ ID NO:58, SEQ ID NO:46, and SEQ ID NO:49, which are at least 55nucleotides in length, which are at least 60 nucleotides in length,which are at least about 65 nucleotides in length, which are at least 70nucleotides in length, which are at least 80 nucleotides in length,which are at least 90 nucleotides in length, which are at least 100nucleotides in length, which are at least 200 nucleotides in length,which are at least 300 nucleotides in length, which are at least 400nucleotides in length, which are at least 500 nucleotides in length,which are at least 600 nucleotides in length, or which preferably arefull-length.

Preferred portions, or fragments, of a feline IL-18, feline caspase-1,canine or feline IL-12 single chain protein of the present inventioninclude at least 15 amino acids, at least 20 amino acids, at least 25amino acids, at least 30 amino acids, at least 35 amino acids, at least40 amino acids, at least 45 amino acids, at least 50 amino acids, atleast 60 amino acids, at least 75 amino acids or at least 100 aminoacids. An IL-18 or IL-12 single chain protein of the present inventioncan include at least a portion of an IL-18 or IL-12 single chain proteinthat is capable of binding to an IL-18 or IL-12 receptor, respectively.These receptors are known to those of skill in the art, and aredescribed in Janeway et al., in Immunobiology, the Immune System inHealth and Disease, Garland Publishing, Inc., NY, 1996 (which isincorporated herein by this reference in its entirety). The IL-18 orIL-12 receptor-binding portion of an IL-18 or IL-12 protein,respectively, can be determined by incubating the protein with anisolated IL-18 or IL-12 receptor, as appropriate, or a cell having anIL-18 or IL-12 receptor on its surface, as appropriate. IL-18 or IL-12protein binding to purified IL-18 or IL-12 receptor, respectively, canbe determined using methods known in the art including Biacore®screening, confocal immunofluorescent microscopy, immunoprecipitation,gel chromatography, determination of inhibition of binding of antibodiesthat bind specifically to the IL-18 or IL-12 binding domain of an IL-18or IL-12 receptor, ELISA using an IL-18 or IL-12 receptor, respectively,labeled with a detectable tag such as an enzyme or chemiluminescent tagor yeast-2 hybrid technology. A caspase-1 protein of the presentinvention can include at least a portion of a caspase-1 protein that iscapable cleaving pro-IL-18 to mature IL-18. The ability of the caspase-1protein to cleave IL-18 can be determined by methods known in the art,including methods such as Biacore® screening, confocal immunofluorescentmicroscopy, immunoprecipitation, gel chromatography, determination ofinhibition of cleavage upon binding of antibodies that bind specificallyto either IL-18 or caspase-1, and enzymatic assays.

The present invention also includes mimetopes of feline IL-18, felinecaspase-1, and canine and/or feline IL-12 single chain proteins of thepresent invention. As used herein, a mimetope of an immunoregulatoryprotein of the present invention refers to any compound that is able tomimic the activity of such a feline IL-18, feline caspase-1, and canineand/or feline IL-12 single chain protein, respectively, often becausethe mimetope has a structure that mimics the particular protein.Mimetopes can be, but are not limited to: peptides that have beenmodified to decrease their susceptibility to degradation such as all-Dretro peptides; anti-idiotypic and/or catalytic antibodies, or fragmentsthereof; non-proteinaceous immunogenic portions of an isolated protein(e.g., carbohydrate structures); and/or synthetic or natural organicmolecules, including nucleic acids. Such mimetopes can be designed usingcomputer-generated structures of proteins of the present invention.Mimetopes can also be obtained by generating random samples ofmolecules, such as oligonucleotides, peptides or other organicmolecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

Furthermore, it is known in the art that there are commerciallyavailable computer programs for determining the degree of similaritybetween two nucleic acid or protein sequences. These computer programsinclude various known methods to determine the percentage identity andthe number and length of gaps between hybrid nucleic acid molecules orproteins. Preferred methods to determine the percent identity amongamino acid sequences and also among nucleic acid sequences includeanalysis using one or more of the commercially available computerprograms designed to compare and analyze nucleic acid or amino acidsequences. These computer programs include, but are not limited to, theSeqLab® Wisconsin Package™ Version 10.0-UNIX sequence analysis software,available from Genetics Computer Group, Madison, Wis.; and DNAsis®sequence analysis software, version 2.0, available from HitachiSoftware, San Bruno, Calif. Such software programs represent acollection of algorithms paired with a graphical user interface forusing the algorithms. The DNAsis version 2.0 software and SeqLabWisconsin Package Version 10.0-UNIX software, for example, employ aparticular algorithm, the Needleman-Wunsch algorithm to performpair-wise comparisons between two sequences to yield a percentageidentity score, see Needleman, S. B. and Wunsch, C.D., 1970, J. Mol.Biol., 48, 443, which is incorporated herein by reference in itsentirety. Such algorithms, including the Needleman-Wunsch algorithm, arecommonly used by those skilled in the nucleic acid and amino acidsequencing art to compare sequences. A preferred method to determinepercent identity among amino acid sequences and also among nucleic acidsequences includes using the Needleman-Wunsch algorithm, available inthe SeqLab Wisconsin Package Version 10.0-UNIX software (hereinafter“SeqLab”), using the Pairwise Comparison/Gap function with thenwsgapdna.cmp scoring matrix, the gap creation penalty and the gapextension penalties set at default values, and the gap shift limits setat maximum (hereinafter referred to as “SeqLab default parameters”). Anadditional preferred method to determine percent identity among aminoacid sequences and also among nucleic acid sequences includes using theHiggins-Sharp algorithm, available in the DNAsis version 2.0 software(hereinafter “DNAsis”), with the gap penalty set at 5, the number of topdiagonals set at 5, the fixed gap penalty set at 10, the k-tuple set at2, the window size set at 5, and the floating gap penalty set at 10. Aparticularly preferred method to determine percent identity among aminoacid sequences and also among nucleic acid sequences includes using theNeedleman-Wunsch algorithm available in the DNAsis version 2.0 software,using the GCG default parameter function.

Another embodiment of the present invention includes a nucleic acidmolecule that is selected from the group consisting of: SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ IDNO:9, SEQ ID NO:41, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:66, and SEQ ID NO:68, and anucleic acid molecule comprising an allelic variant of a nucleic acidmolecule comprising any of said nucleic acid sequences. An allelicvariant of a feline and/or canine nucleic acid molecule of the presentinvention, including the particular SEQ ID NO's cited herein, is a genethat occurs at essentially the same locus (or loci) in the genome as thegene including the particular SEQ ID NO's cited herein, but which, dueto natural variations caused by, for example, mutation or recombination,has a similar but not identical sequence. Also included in the termallelic variant are allelic variants of cDNAs derived from such genes.Because natural selection typically selects against alterations thataffect function, allelic variants usually encode proteins having similaractivity to that of the protein encoded by the gene to which they arebeing compared. Allelic variants of genes or nucleic acid molecules canalso comprise alterations in the 5′ or 3′ untranslated regions of thegene (e.g., in regulatory control regions), or can involve alternativesplicing of a nascent transcript, thereby bringing alternative exonsinto juxtaposition. Allelic variants are well known to those skilled inthe art and would be expected to be found within a given animal, sincethe respective genomes are diploid, and sexual reproduction will resultin the reassortment of alleles. As such, a nucleic acid molecule of thepresent invention can be an allelic variant that includes a similar butnot identical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:66, and SEQ ID NO:68, and/or any other nucleic acidmolecule cited herein.

In another embodiment of the present invention, a nucleic acid moleculeof the invention is selected from the group consisting of (a) a nucleicacid molecule comprising a nucleic acid sequence that encodes a proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:44, SEQ IDNO:62, and SEQ ID NO:67, and (b) a nucleic acid molecule comprising anallelic variant of a nucleic acid molecule encoding a protein having anyof said amino acid sequences of (a).

Another embodiment of the present invention includes feline IL-18nucleic acid molecules of the present invention, wherein said nucleicacid molecules encode a protein having a function selected from thegroup consisting of (i) eliciting an immune response against an IL-18protein having an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12; (ii)selectively binding to an antibody raised against an IL-18 proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12, and (iii)exhibiting IL-18 activity. Methods to elicit an immune response and todetermine whether an antibody can selectively bind to a particularprotein or antigen are known in the art, see, for example, Harlow, etal. (1988) Antibodies, a Laboratory Manual, Cold Spring Harbor LabsPress; Harlow, et al. is incorporated by reference herein in itsentirety. Methods to determine whether an IL-18 protein has IL-18activity are known in the art, and include determining whether IL-18 hasthe activity of stimulating T cells to produce interferon gamma (IFN-γ).

Another embodiment of the present invention includes caspase-1 nucleicacid molecules of the present invention that encode a protein having afunction selected from the group consisting of (i) eliciting an immuneresponse against a caspase-1 protein having an amino acid sequenceselected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ IDNO:21, and SEQ ID NO:24, (ii) selectively binding to an antibody raisedagainst caspase-1 protein having an amino acid sequence selected fromthe group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, andSEQ ID NO:24, and (iii) exhibiting caspase-1 activity. Methods to elicitan immune response and to determine whether an antibody can selectivelybind to a particular protein or antigen are known in the art, see, forexample, Harlow, et al. (1988) Antibodies, a Laboratory Manual, ColdSpring Harbor Labs Press; Harlow, et al. is incorporated by referenceherein in its entirety. Methods to determine whether a caspase-1 proteinhas caspase-1 activity are known in the art, and include, for example,determining if the caspase-1 protein has the ability to cleave theprecursor form of IL-18 resulting in a biologically active IL-18.

Another embodiment of the present invention includes canine and felineIL-12 single chain nucleic acid molecules of the present invention,wherein a said nucleic acid molecule encodes a protein having a functionselected from the group consisting of (i) eliciting an immune responseagainst an IL-12 protein having an amino acid selected from the groupconsisting of SEQ ID NO:39, SEQ ID NO:44, SEQ ID NO:62, and SEQ IDNO:67, (ii) selectively binding to an antibody raised against an IL-12protein having an amino acid sequence selected from the group consistingfrom the group of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQID NO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12 activity.Methods to elicit an immune response and to determine whether anantibody can selectively bind to a particular protein or antigen areknown in the art, see, for example, Harlow, et al. (1988) Antibodies, aLaboratory Manual, Cold Spring Harbor Labs Press; Harlow, et al. isincorporated by reference herein in its entirety. Methods to determinewhether an IL-12 protein has IL-12 activity are known in the art, andinclude determining if IL-12 has the activity of stimulating T cells toproduce interferon gamma (IFN-γ).

A preferred nucleic acid molecule of the present invention includes anucleic acid molecule selected from the group consisting ofnFeIL-12p40-N₉₈₅, nFeIL-12p40₉₈₇, nFeIL-12p40₉₂₁, nFeIL-12p35₆₆₆,nFeIL-12p35-N₅₆₁, nFeIL-12p35₅₉₁, nCaIL-12p35₆₆₆, nCaIL-12p35₁₄₅₅,nCaIL-12p35₅₉₁, nCaIL-12p40₂₂₆₇, nCaIL-12p40₉₂₁, nCaIL-12p40₉₈,nFeIL-12₁₅₉₉, nFeIL-12₁₅₃₃, nCaIL-12₁₅₉₉, and nCaIL-12₁₅₃₃.

Another embodiment of the present invention includes an isolated nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule having a nucleic acid sequence encoding an IL-18 proteinselected from the group consisting of: a protein selected from the groupconsisting of (a) a protein having an amino acid sequence that is atleast 92 percent identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ IDNO:12, and (b) a protein comprising a fragment of a protein of (a),wherein said fragment is at least 30 amino acids in length; and aprotein comprising at least 25 contiguous amino acids identical insequence to at least 25 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12. Preferred IL-18 proteins include proteins thatare at least about 90 percent identical, preferably at least about 92percent identical, preferably at least about 94 percent identical,preferably at least about 96 percent identical, and even more preferablyat least about 98 percent identical to SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12 or fragments thereof. Preferred fragments ofIL-18 proteins include fragments of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12 that are at least about 20 amino acids in length,at least about 30 amino acids in length, at least about 40 amino acidsin length, at least about 50 amino acids in length, preferably at leastabout 75 amino acids in length, preferably at least about 100 aminoacids in length, and more preferably are full-length. Preferred IL-18proteins also include proteins that comprise at least 15 contiguousamino acids identical in sequence to at least 15 contiguous amino acids;at least 20 contiguous amino acids identical in sequence to at least 20contiguous amino acids, preferably about 30 contiguous amino acidsidentical in sequence to at least 30 contiguous amino acids, preferablyabout 50 contiguous amino acids identical in sequence to at least 50contiguous amino acids, preferably about 75 contiguous amino acidsidentical in sequence to at least 75 contiguous amino acids, preferablyabout 100 contiguous amino acids identical in sequence to at least 100contiguous amino acids, and most preferably a full-length proteinidentical in sequence to a full-length protein of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12.

Another embodiment of the present invention includes an isolated nucleicacid molecule selected from the group consisting of: a nucleic acidmolecule having a nucleic acid sequence encoding caspase proteinselected from the group consisting of: a protein selected from the groupconsisting of (a) a protein having an amino acid sequence that is atleast 85 percent identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ IDNO:24, and (b) a protein comprising a fragment of a protein of (a),wherein said fragment is at least 30 amino acids in length; and aprotein comprising at least 25 contiguous amino acids identical insequence to at least 25 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12. Preferred caspase-1 proteins include proteinsthat are at least about 85 percent identical, at least about 87 percentidentical, preferably at least about 90 percent identical, preferably atleast about 93 percent identical, more preferably at least about 95percent identical, and even more preferably about 98 percent identicalto SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:2 1, and SEQ ID NO:24 orfragments thereof. Preferred fragments of caspase-1 proteins includefragments of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24include fragments that are at least about 20 amino acids in length, atleast about 30 amino acids in length, at least about 40 amino acids inlength, at least about 50 amino acids in length, at least about 60 aminoacids in length, preferably at least about 75 amino acids in length,preferably at least about 100 amino acids in length, and more preferablyare full-length. Preferred caspase-1 proteins also include proteins thatcomprise at least 25 contiguous amino acids identical in sequence to atleast 25 contiguous amino acids; at least 20 contiguous amino acidsidentical in sequence to at least 20 contiguous amino acids, preferablyabout 30 contiguous amino acids identical in sequence to at least 30contiguous amino acids, preferably about 50 contiguous amino acidsidentical in sequence to at least 50 contiguous amino acids, preferablyabout 75 contiguous amino acids identical in sequence to at least 75contiguous amino acids, preferably about 100 contiguous amino acidsidentical in sequence to at least 100 contiguous amino acids, and mostpreferably a full-length protein identical in sequence to a full-lengthprotein of an amino acid sequence selected from the group consisting ofSEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24.

Another embodiment of the present invention includes a nucleic acidmolecule having a nucleic acid sequence encoding an IL-12 single chainprotein comprising an IL-12p40 subunit domain linked to a IL-12p35subunit domain, wherein said p40 subunit domain is selected from thegroup consisting of: (i) a p40 subunit protein having an amino acidsequence that is at least 84 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30,(ii) a p40 subunit protein comprising a fragment of a protein of (i),wherein said fragment is at least 30 amino acids in length, and (iii) ap40 subunit protein comprising at least 23 contiguous amino acidsidentical in sequence to at least 23 contiguous amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:27 and SEQID NO:30, and wherein said p35 domain is selected from the groupconsisting of (i) a p35 subunit protein having an amino acid sequencethat is at least 84 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:33 and SEQ ID NO:36, (ii) a p35subunit protein comprising a fragment of a protein of (i), wherein saidfragment is at least 30 amino acids in length, and (iii) a p35 subunitprotein comprising at least 23 contiguous amino acids identical insequence to at least 23 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36.Preferred p40 subunit proteins and/or p35 subunit proteins includeproteins that are at least about 84 percent identical, preferably atleast about 87 percent identical, preferably at least about 90 percentidentical, and even more preferably at least about 95 percent identicalto SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36 orfragments thereof. Preferred fragments of IL-12 single chain proteinsinclude fragments of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQID NO:36 include fragments that are at least about 30 amino acids inlength, at least about 40 amino acids in length, at least about 50 aminoacids in length, at least about 60 amino acids in length, preferably atleast about 75 amino acids in length, preferably at least about 100amino acids in length, and more preferably are full-length. PreferredIL-12 single chain proteins also include proteins that comprise at least23 contiguous amino acids identical in sequence to at least 23contiguous amino acids, preferably about 30 contiguous amino acidsidentical in sequence to at least 30 contiguous amino acids, preferablyabout 50 contiguous amino acids identical in sequence to at least 50contiguous amino acids, preferably about 75 contiguous amino acidsidentical in sequence to at least 75 contiguous amino acids, preferablyabout 100 contiguous amino acids identical in sequence to at least 100contiguous amino acids, and most preferably a full-length proteinidentical in sequence to a full-length protein of an amino acid sequenceselected from the group consisting of SEQ ID NO:27, SEQ ID NO:30, SEQ IDNO:33, and SEQ ID NO:36.

Another embodiment of the present invention includes a nucleic acidmolecule having a nucleic acid sequence encoding an IL-12 single chainprotein comprising an IL-12p40 subunit domain linked to a IL-12p35subunit domain, wherein said p40 subunit domain is selected from thegroup consisting of: (i) a p40 subunit protein having an amino acidsequence that is at least 84 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:53 and SEQ ID NO:59,(ii) a p40 subunit protein comprising a fragment of a protein of (i),wherein said fragment is at least 40 amino acids in length, and (iii) ap40 subunit protein comprising at least 31 contiguous amino acidsidentical in sequence to at least 31 contiguous amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:53 and SEQID NO:59, and wherein said p35 domain is selected from the groupconsisting of (i) a p35 subunit protein having an amino acid sequencethat is at least 84 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35subunit protein comprising a fragment of a protein of (i), wherein saidfragment is at least 40 amino acids in length, and (iii) a p35 subunitprotein comprising at least 31 contiguous amino acids identical insequence to at least 31 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50.Preferred p40 subunit proteins and/or p35 subunit proteins includeproteins that are at least about 84 percent identical, preferably atleast about 87 percent identical, preferably at least about 90 percentidentical, and even more preferably at least about 95 percent identicalto SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59 orfragments thereof. Preferred fragments of IL-12 single chain proteinsinclude fragments of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQID NO:36 include fragments that are at least about 40 amino acids inlength, at least about 50 amino acids in length, at least about 60 aminoacids in length, at least about 70 amino acids in length, preferably atleast about 80 amino acids in length, preferably at least about 100amino acids in length, and more preferably are full-length. PreferredIL-12 single chain proteins also include proteins that comprise at least31 contiguous amino acids identical in sequence to at least 31contiguous amino acids, preferably about 35 contiguous amino acidsidentical in sequence to at least 35 contiguous amino acids, preferablyabout 50 contiguous amino acids identical in sequence to at least 50contiguous amino acids, preferably about 75 contiguous amino acidsidentical in sequence to at least 75 contiguous amino acids, preferablyabout 100 contiguous amino acids identical in sequence to at least 100contiguous amino acids, and most preferably a full-length proteinidentical in sequence to a full-length protein of an amino acid sequenceselected from the group consisting of SEQ ID NO:47, SEQ ID NO:50, SEQ IDNO:53, and SEQ ID NO:59.

Another embodiment of the present invention includes a nucleic acidmolecule comprising a nucleic acid sequence fully complementary to thecoding strand of any of the nucleic acid molecules of the presentinvention. Another embodiment of the present invention includes anucleic acid molecule that comprises a nucleic acid sequence thatencodes a protein selected from the group consisting of an IL-18protein, a caspase-1 protein, and an IL-12 single chain protein.

Another embodiment of the present invention includes a nucleic acidmolecule that is selected from the group consisting of a nucleic acidmolecule comprising a nucleic acid sequence encoding a proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, SEQID NO;18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:44, SEQ IDNO:62, and SEQ ID NO:67; and a nucleic acid molecule comprising anallelic variant of a nucleic acid molecule encoding a protein having anyof said nucleic acid molecules set forth in this paragraph. In anotherembodiment, a nucleic acid molecule encoding an IL-12 single chainprotein of the present invention further comprises a nucleic acidmolecule encoding a linker.

The present invention also includes oligonucleotides, recombinantmolecules, recombinant viruses and recombinant cells comprising suchnucleic acid molecules and methods to produce such nucleic acidmolecules, oligonucleotides, recombinant molecules, recombinant virusesand recombinant cells.

Knowing the nucleic acid sequences of certain nucleic acid molecules ofthe present invention allows one skilled in the art to, for example, (a)make copies of those nucleic acid molecules, (b) obtain nucleic acidmolecules including at least a portion of such nucleic acid molecules,e.g., nucleic acid molecules including full-length genes, full-lengthcoding regions, regulatory control sequences, truncated coding regions,and Ĉ) obtain other nucleic acid molecules. Such nucleic acid moleculescan be obtained in a variety of ways including screening appropriateexpression libraries with antibodies of the present invention;traditional cloning techniques using oligonucleotide probes of thepresent invention to screen appropriate libraries; and PCR amplificationof appropriate libraries or DNA using oligonucleotide primers of thepresent invention. A preferred library to screen or from which toamplify nucleic acid molecules is a feline or canine mast library or afeline or canine peripheral blood mononuclear cell library. Techniquesto clone and amplify genes are disclosed, for example, in Sambrook etal., ibid.

Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a larger nucleic acidmolecule of the present invention, typically from about 12 to 15 toabout 17 to 18 nucleotides depending on the GC/AT content. The presentinvention includes oligonucleotides that can be used as, for example,probes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit protein production oractivity, e.g., as antisense-, triplex formation-, ribozyme- and/or RNAdrug-based reagents. The present invention also includes the use of sucholigonucleotides to protect animals from disease using one or more ofsuch technologies. Appropriate oligonucleotide-containing therapeuticcompositions can be administered to an animal using techniques known tothose skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulating of the nucleicacid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the nucleic acid molecule is able to beexpressed when transformed into a host cell. As used herein, anexpression vector is a DNA or RNA vector that is capable of transforminga host cell and of effecting expression of a specified nucleic acidmolecule. Preferably, the expression vector is also capable ofreplicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction, i.e., direct gene expression, in recombinant cells of thepresent invention, including in bacterial, fungal, insect, other animal,and plant cells. Preferred expression vectors of the present inventioncan direct gene expression in bacterial, yeast, insect and mammaliancells, and more preferably in the cell types disclosed herein.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences that control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those that control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, or insect andmammalian cells, such as, but not limited to, tac, lac, trp, trc,oxy-pro, omp/lpp, rrnB, bacteriophage lambda, such as lambda P_(L) andlambda p_(R) and fusions that include such promoters, bacteriophage T7,T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01,metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirussubgenomic promoter, antibiotic resistance gene, baculovirus, Heliothiszea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, otherpoxvirus, adenovirus, cytomegalovirus, such as immediate early promoter,simian virus 40, retrovirus, actin, retroviral long terminal repeat,Rous sarcoma virus, heat shock, phosphate and nitrate transcriptioncontrol sequences as well as other sequences capable of controlling geneexpression in prokaryotic or eukaryotic cells. Additional suitabletranscription control sequences include tissue-specific promoters andenhancers as well as lymphokine-inducible promoters, e.g., promotersinducible by interferons or interleukins.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transfercells are disclosed herein. A recombinant cell is preferably produced bytransforming a host cell with one or more recombinant molecules, eachcomprising one or more nucleic acid molecules of the present inventionoperatively linked to an expression vector containing one or moretranscription control sequences, examples of which are disclosed herein.

Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. A recombinant cell may remainunicellular or may grow into a tissue, organ or a multicellularorganism. It is to be noted that a cell line refers to any recombinantcell of the present invention that is not a transgenic animal.Transformed nucleic acid molecules of the present invention can remainextrachromosomal or can integrate into one or more sites within achromosome of the transformed, i.e., recombinant, cell in such a mannerthat their ability to be expressed is retained. Preferred nucleic acidmolecules with which to transform a cell include nucleic acid moleculesdisclosed herein. Particularly preferred nucleic acid molecules withwhich to transform a cell include nFeIL-18-N₅₁₄, nFeIL-18-C₅₀₂,nFeIL-18₆₀₇, nFeIL-18₅₇₆, nFeIL-18₄₇₁, nFeCasp-1₁₂₃₃, nFeCasp-1-N₅₂₆,nFeCasp-1-C₅₀₀, nFeCasp-1₁₂₃₀, nFeIL-12p40-N₉₈₅, nFeIL-12p40₉₈₇,nFeIL-12p40₉₂₁, nFeIL-12p35₆₆₆, nFeIL-12p35-N₅₆₁, nFeIL-12p35₅₉₁,nCaIL-12p35₆₆₆, nCaIL-12p35₁₄₅₅, nCaIL-12p35₅₉₁, nCaIL-12p40₂₂₆₇,nCaIL-12p40₉₂₁, and nCaIL-12p40_(987.,)nCaIL-12₁₅₉₉, nCaIL-12₁₅₃₃,nFeIL-12₁₅₉₉, and nFeIL-12₁₅₃₃.

Recombinant molecules of the present invention may also (a) containsecretory signals, i.e., signal segment nucleic acid sequences, toenable an expressed protein of the present invention to be secreted fromthe cell that produces the protein and/or (b) contain fusion sequenceswhich lead to the expression of nucleic acid molecules of the presentinvention as fusion proteins. Examples of suitable signal segmentsinclude any signal segment capable of directing the secretion of aprotein of the present invention. Preferred signal segments include, butare not limited to, tissue plasminogen activator (t-PA), interferon,interleukin, growth hormone, histocompatibility and viral envelopeglycoprotein signal segments. Suitable fusion segments encoded by fusionsegment nucleic acids are disclosed herein. In addition, a nucleic acidmolecule of the present invention can be joined to a fusion segment thatdirects the encoded protein to the proteosome, such as a ubiquitinfusion segment. Eukaryotic recombinant molecules may also includeintervening and/or untranslated sequences surrounding and/or within thenucleic acid sequences of nucleic acid molecules of the presentinvention.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule, e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins. Host cells of the present invention either can beendogenously, i.e., naturally, capable of producing proteins of thepresent invention or can be capable of producing such proteins afterbeing transformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fungal, including yeast, insect, and other animal and plantcells. Preferred host cells include bacterial, mycobacterial, yeast,insect and mammalian cells. More preferred host cells includeSalmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Pichia,Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells,MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandellfeline kidney cell line), CV-1 cells (African monkey kidney cell lineused, for example, to culture raccoon poxvirus), COS (e.g., COS-7)cells, and Vero cells. Particularly preferred host cells are Escherichiacoli, including E. coli K-12 derivatives; Salmonella typhi; Salmonellatyphimurium, including attenuated strains such as UK-1 _(χ)3987 andSR-11 _(χ)4072; Pichia; Spodoptera frugiperda; Trichoplusia ni; BHKcells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; andnon-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).Additional appropriate mammalian cell hosts include other kidney celllines, other fibroblast cell lines, e.g., human, murine or chickenembryo fibroblast cell lines, myeloma cell lines, Chinese hamster ovarycells, mouse NIH/3T3 cells, LMTK³¹ cells and/or HeLa cells.

Recombinant cells of the present invention can also be co-transformedwith one or more recombinant molecules including IL-18, caspase-1, IL-12single chain nucleic acid molecules encoding one or more proteins of thepresent invention and one or more other nucleic acid molecules encodingother compounds.

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals, e.g., promoters,operators, enhancers, substitutions or modifications of translationalcontrol signals, e.g., ribosome binding sites, Shine-Dalgarno sequences,modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant enzyme productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing nucleic acid molecules encoding such a protein.

Isolated feline IL-18, feline caspase-1, feline and/or canine IL-12single chain proteins of the present invention can be produced in avariety of ways, including production and recovery of natural proteins,production and recovery of recombinant proteins, and chemical synthesisof the proteins. In one embodiment, an isolated protein of the presentinvention is produced by culturing a cell capable of expressing theprotein under conditions effective to produce the protein, andrecovering the protein. A preferred cell to culture is a recombinantcell of the present invention. Effective culture conditions include, butare not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce a protein ofthe present invention. Such a medium typically comprises an aqueousmedium having assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. Cells of the present invention can be cultured in conventionalfermentation bioreactors, shake flasks, test tubes, microtiter dishes,and petri plates. Culturing can be carried out at a temperature, pH andoxygen content appropriate for a recombinant cell. Such culturingconditions are within the expertise of one of ordinary skill in the art.Examples of suitable conditions are included in the Examples section.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane. The phrase recovering the protein, as well as similar phrases,refers to collecting the whole fermentation medium containing theprotein and need not imply additional steps of separation orpurification. Proteins of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.Proteins of the present invention are preferably retrieved insubstantially pure form. As used herein, substantially pure refers to apurity that allows for the effective use of the protein as a therapeuticcomposition or diagnostic. A therapeutic composition for animals, forexample, should exhibit no substantial toxicity.

The present invention also includes isolated, i.e., removed from theirnatural milieu, antibodies that selectively bind to proteins of thepresent invention or a mimetope thereof, e.g., anti-feline IL-18, felinecaspase-1, feline and canine IL-12 single antibodies. As used herein,the term selectively binds to a protein refers to the ability ofantibodies of the present invention to preferentially bind to specifiedproteins and mimetopes thereof of the present invention. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays, e.g., ELISA, immunoblot assays, etc.; see, for example,Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, aLaboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid.,is incorporated herein by reference in its entirety. For example, ananti-feline IL-18 antibody of the present invention preferablyselectively binds to a feline IL-18 protein in such a way as to inhibitthe function of that protein.

The antibodies of the present invention bind to the proteins of thepresent invention, but not to similar proteins of other species. Forinstance, the antibodies that specifically bind feline IL-18 do not bindcanine IL-18.

Isolated antibodies of the present invention can include antibodies inserum, or antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal, orcan be functional equivalents such as antibody fragments andgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide or mimetope of the present invention to produce theantibodies and (b) recovering the antibodies. In another method,antibodies of the present invention are produced recombinantly usingtechniques as heretofore disclosed to produce proteins of the presentinvention. Antibodies raised against defined proteins or mimetopes canbe advantageous because such antibodies are not substantiallycontaminated with antibodies against other substances that mightotherwise cause interference in a diagnostic assay or side effects ifused in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) to evaluate the immune status in felids andcanids with diseases such as allergy, cancer and pathogen infections.Furthermore, antibodies of the present invention can be used to targetcytotoxic agents to cells. Targeting can be accomplished by conjugating,i.e., stably joining, such antibodies to the cytotoxic agents usingtechniques known to those skilled in the art. Suitable cytotoxic agentsare known to those skilled in the art. Furthermore, antibodies of thepresent invention can be used to detect for example, feline IL-18,caspase-1, canine IL-12 single chain, and/or feline IL-12 single chainin a putative IL-18, caspase-1, canine IL-12 single chain, and/or felineIL-12 single chain containing biological sample, by contacting theputative IL-18, caspase-1, canine IL-12 single chain, and/or felineIL-12 single chain containing biological sample with the appropriateanti-IL-18, caspase-1, canine IL-12 single chain, and/or feline IL-12single chain antibodies under conditions suitable for formation of anantigen-antibody complex, and then detecting said complex. Methods todetect said method are known to those skilled in the art and arecontained herein.

The present invention includes proteins comprising SEQ ID NO:2, SEQ IDNO:5, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ IDNO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ IDNO:36, SEQ ID NO:39, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ IDNO:53, SEQ ID NO:59, SEQ ID NO:62, and SEQ ID NO:67 as well as nucleicacid molecules encoding such proteins.

Preferred feline IL-18 proteins of the present invention includePFeIL-18-N₁₃₃, PFeIL-18-C₁₅₄, PFeIL-18₁₉₂, and/or PFeIL-18₁₅₇. In oneembodiment, a preferred feline IL-18 protein of the present inventionhas an amino acid sequence that includes SEQ ID NO:2, SEQ ID NO:5, SEQID NO:8, and/or SEQ ID NO:12 and is preferably encoded by a nucleic acidmolecule having nucleic acid sequences SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:7, SEQ ID NO:9 and/or SEQ ID NO:11. Such proteins are preferablyencoded by a nucleic acid molecule comprising nFeIL-18-N₅₁₄,nFeIL-18-C₅₀₂, nFeIL-18₆₀₇, nFeIL-18₅₇₆, and/or nFeIL-18₄₇₁.

Preferred feline caspase-1 proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nFeCasp-1₁₂₃₃,nFeCasp-1-N₅₂₆, nFeCasp-1-C₅₀₀, and/or nFeCasp-1₁₂₃₀. Preferred felinecaspase-1 proteins are PFeCasp-1₄₁₀, PFeCasp-1-N₁₆₉, and/orPFeCasp-1-C₁₂₀. In one embodiment, a preferred feline caspase-1 proteinof the present invention is encoded by SEQ ID NO:14, SEQ ID NO:17, SEQID NO:20, and/or SEQ ID NO:23, and, as such, has an amino acid sequencethat includes SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21 and/or SEQ IDNO:24.

Preferred canine and feline IL-12 proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnFeIL-12₁₅₉₉, nFeIL-12₁₅₃₃, nCaIL-12₁₅₉₉, and/or nCaIL-12₁₅₃₃. Preferredfeline and canine IL-12 proteins are nFeIL-12p40-N₉₈₅, nFeIL-12p40₉₈₇,nFeIL-12p40₉₂₁, nFeIL-12p35₆₆₆, nFeIL-12p 35-N₅₆₁, nFeIL-12p35₅₉₁,nCaIL-12p35₆₆₆, nCaIL-12p35₁₄₅₅, nCaIL-12p35₅₉₁, nCaIL-12p 40₂₂₆₇,nCaIL-12p40₉₂₁, and nCaIL-12p40₉₈₇. In one embodiment, a preferredcanine and feline IL-12 single chain protein of the present invention isencoded by SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:61, and/or SEQ IDNO:66, and, as such, has an amino acid sequence that includes SEQ IDNO:39, SEQ ID NO:44, SEQ ID NO:62, and/or SEQ ID NO:67.

More preferred canine and feline IL-12 single chain proteins of thepresent invention include proteins encoded by a nucleic acid moleculecomprising nFeIL-12p40-N₉₈₅, nFeIL-12p40₉₈₇, nFeIL-12p40₉₂₁,nFeIL-12p35₆₆₆, nFeIL-12p35-N₅₆₁, nFeIL-12p35₅₉₁, nCaIL-12p35₆₆₆,nCaIL-12p35₁₄₅₅, nCaIL-12p35₅₉₁, nCaIL-12p40₂₂₆₇, nCaIL-12p40₉₂₁,nCaIL-12p40₉₈₇, nCaIL-12₁₅₃₃, nCaIL-12₁₅₉₉, nFeIL-12₁₅₃₃, andnFeIL-12₁₅₉₉. Preferred feline and canine IL-12 single chain proteinscomprise PFeIL-12p40-N₃₂₈, PFeIL-12p40₃₂₉, PFeIL-12p40₃₀₇,PFeIL-12p35₂₂₂, PFeIL-12p35-N₁₈₇, PFeIL-12p35₁₉₇, PCaIL-12p35₂₂₂,PCaIL-12p35₁₉₇, PCaIL-12p40₃₀₇, PCaIL-12p40₃₂₉, PFeIL-12₅₃₃,PFeIL-12₅₁₁, PCaIL-12₅₃₃, and PCaIL-12₅₁₁. In one embodiment, apreferred canine and feline IL-12 single chain protein of the presentinvention is encoded by a nucleic acid comprising SEQ ID NO:28, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:46, SEQ ID NO:49, SEQ IDNO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:101, SEQ ID NO:104, SEQ IDNO:107, SEQ ID NO:38, SEQ ID NO:43, SEQ ID NO:61, SEQ ID NO:66, and, assuch, has an amino acid sequence that includes SEQ ID NO:27, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ IDNO:53, and SEQ ID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:62, and/or SEQ ID NO:67.

As used herein, an isolated protein of the present invention can be afull-length protein or any homolog of such a protein. An isolatedprotein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to bind to areceptor or a protein. Examples of protein homologs of the presentinvention include proteins of the present invention in which amino acidshave been deleted (e.g., a truncated version of the protein, such as apeptide), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol) such that the protein homolog includes atleast one epitope capable of eliciting an immune response against theparent protein, of binding to an antibody directed against the parentprotein and/or of binding to the parent's receptor, where the termparent refers to the longer and/or full-length protein that the homologis derived from. That is, when the homolog is administered to an animalas an immunogen, using techniques known to those skilled in the art, theanimal will produce an immune response against at least one epitope ofan immunoregulatory protein of the present invention, depending uponwhich protein is administered to an animal. The ability of a protein toeffect an immune response can be measured using techniques known tothose skilled in the art.

Homologs of proteins of the present invention can be the result ofnatural allelic variation, including natural mutation. Protein homologsof the present invention can also be produced using techniques known inthe art including, but not limited to, direct modifications to theprotein and/or modifications to the gene encoding the protein using, forexample, classic or recombinant DNA techniques to effect random ortargeted mutagenesis.

One embodiment of the present invention is an IL-18 protein selectedfrom the group consisting of: (i) a protein having an amino acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8 and SEQ ID NO:12; and (ii) a protein encoded by an allelicvariant of a nucleic acid molecule encoding a protein selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ IDNO:12. Another embodiment is a caspase-1 protein selected from the groupconsisting of: (i) a protein having an amino acid sequence selected fromthe group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, andSEQ ID NO:24; and (ii) a protein encoded by an allelic variant of anucleic acid molecule encoding a protein selected from the groupconsisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ IDNO:24. Yet another embodiment is a feline IL-12 single chain proteinselected from the group consisting of: (i) a protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:38, andSEQ ID NO:44; and (ii) a protein encoded by an allelic variant of anucleic acid molecule encoding a protein selected from the groupconsisting of SEQ ID NO:38, and SEQ ID NO:44; or a canine Il-12 singlechain protein selected from the group consisting of: (i) a proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:62 and SEQ ID NO:67; and (ii) a protein encoded by an allelicvariant of a nucleic acid molecule encoding a protein selected from thegroup consisting of SEQ ID NO:62 and SEQ ID NO:67.

One embodiment of the present invention includes an isolated IL-18protein selected from the group consisting of (i) an isolated protein ofat least 25 amino acids in length, wherein said protein has an at least25 contiguous amino acid region identical in sequence to a 25 contiguousamino acid region selected from the group consisting of SEQ ID NO:2, SEQID NO:5, SEQ ID NO:8, and SEQ ID NO:12; and (ii) an isolated proteinhaving an amino acid sequence that is at least 92 percent identical toan amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12, and a fragment thereofof at least 30 nucleotides. Preferred proteins have an at least 15contiguous amino acid region identical with a 15 contiguous amino acidregion, an at least 20 contiguous amino acid region identical with a 20contiguous amino acid region, an at least 30 contiguous amino acidregion identical with a 30 contiguous amino acid region, an at least 40contiguous amino acid region identical with a 40 contiguous amino acidregion, an at least 50 contiguous amino acid region contiguous with a 50contiguous amino acid region, an at least 75 contiguous amino acidregion contiguous with a 75 contiguous amino acid region, preferably anat least 100 contiguous amino acid region contiguous with a 100contiguous amino acid region, and most preferably a full-length proteinidentical in sequence to a full-length protein of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, and SEQ ID NO:12. In another embodiment, preferred proteins havean amino acid sequence that is at least 90 percent identical, at least92 percent identical, preferably at least 94 percent identical,preferably at least 96 percent identical, and even more preferably atleast about 98 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQID NO:12, and a fragment thereof of at least 20 amino acids, at least 30amino acids, at least 50 amino acids, at least 75 amino acids,preferably at least 100 amino acids, and more preferably a full-lengthprotein.

In a preferred embodiment, IL-18 proteins of the present invention has afunction selected from the group consisting of: (i) eliciting an immuneresponse against an IL-18 protein having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, andSEQ ID NO:12, (ii) selectively binding to an antibody raised against anIL-18 protein having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12,and (iii) exhibiting IL-18 activity.

One embodiment of the present invention includes an isolated caspase-1protein selected from the group consisting of (i) an isolated protein ofat least 25 amino acids in length, wherein said protein has an at least25 contiguous amino acid region identical in sequence to a 25 contiguousamino acid region selected from the group consisting of SEQ ID NO:15,SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24; and (ii) an isolatedprotein having an amino acid sequence that is at least 85 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, and afragment thereof of at least 30 nucleotides. Preferred proteins have anat least 25 contiguous amino acid region identical with a 25 contiguousamino acid region, an at least 20 contiguous amino acid region identicalwith a 20 contiguous amino acid region, an at least 30 contiguous aminoacid region identical with a 30 contiguous amino acid region, an atleast 40 contiguous amino acid region identical with a 40 contiguousamino acid region, an at least 50 contiguous amino acid regioncontiguous with a 50 contiguous amino acid region, an at least 75contiguous amino acid region contiguous with a 75 contiguous amino acidregion, preferably an at least 100 contiguous amino acid regioncontiguous with a 100 contiguous amino acid region, and most preferablya full-length protein identical in sequence to a full-length protein ofan amino acid sequence selected from the group consisting of SEQ IDNO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24. In anotherembodiment, preferred proteins have an amino acid sequence that is atleast 85 percent identical, at least 88 percent identical, preferably atleast 90 percent identical, and more preferably at least about 95percent identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ IDNO:24, and a fragment thereof of at least 30 amino acids, at least 50amino acids, at least 75 amino acids, preferably at least 100 aminoacids, and more preferably a full-length protein.

In a preferred embodiment, a caspase protein of the present inventionhas a function selected from the group consisting of: (i) eliciting animmune response against a caspase-1 protein having an amino acidsequence selected from the group consisting of SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, and SEQ ID NO:24, (ii) selectively binding to anantibody raised against a caspase-1 protein having an amino acidsequence selected from the group consisting of SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:21, and SEQ ID NO:24, and (iii) exhibitingcaspase-1activity.

One embodiment of the present invention includes an isolated IL-12single chain protein comprising an IL-12 p40 subunit domain linked to anIL-12 p35 subunit domain, wherein said p40 subunit domain is selectedfrom the group consisting of (i) a p40 subunit protein having an aminoacid sequence that is at least 84 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:27 and SEQ IDNO:30, (ii) a p40 subunit protein comprising a fragment of a protein of(i), wherein said fragment is at least 30 amino acids in length, and(iii) a p40 subunit protein comprising at least 23 contiguous aminoacids identical in sequence to at least 23 contiguous amino acids of anamino acid sequence selected from the group consisting of SEQ ID NO:27and SEQ ID NO:30. The p35 subunit is preferably selected from the groupconsisting of (i) a p35 subunit protein having an amino acid sequencethat is at least 84 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:33 and SEQ ID NO:36, (ii) a p35subunit protein comprising a fragment of a protein of (i), wherein saidfragment is at least 30 amino acids in length, and (iii) a p35 subunitprotein comprising at least 23 contiguous amino acids identical insequence to at least 23 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36.Preferred amino acid sequences have an at least 23 contiguous amino acidregion identical with a 23 contiguous amino acid region, an at least 30contiguous amino acid region identical with a 30 contiguous amino acidregion, an at least 40 contiguous amino acid region identical with a 40contiguous amino acid region, an at least 50 contiguous amino acidregion contiguous with a 50 contiguous amino acid region, an at least 75contiguous amino acid region contiguous with a 75 contiguous amino acidregion, preferably an at least 100 contiguous amino acid regioncontiguous with a 100 contiguous amino acid region, and most preferablya full-length protein identical in sequence to a full-length protein ofan amino acid sequence selected from the group consisting of SEQ IDNO:27 and SEQ ID NO:30 In another embodiment, preferred proteins have anamino acid sequence that is at least 84 percent identical, at least 86percent identical, at least 88 percent identical, preferably at least 90percent identical, and more preferably at least about 95 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:27, SEQ ID NO:30, and a fragment thereof of at least 30amino acids, at least 50 amino acids, at least 75 amino acids,preferably at least 100 amino acids, and more preferably a full-lengthprotein.

In a preferred embodiment, an IL-12 single chain protein of the presentinvention has a function selected from the group consisting of: (i)eliciting an immune response against an IL-12 protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:27, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ IDNO:53, and SEQ ID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:62, and/or SEQ ID NO:67; (ii)selectively binding to an antibody raised against an IL-12 proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ IDNO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:62, and/or SEQ IDNO:67; and (iii) exhibiting IL-12 activity.

One embodiment of the present invention includes an isolated IL-12single chain protein comprising an IL-12 p40 subunit domain linked to anIL-12 p35 subunit domain, wherein said p40 subunit domain is selectedfrom the group consisting of (i) a p40 subunit protein having an aminoacid sequence that is at least 84 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:53 and SEQ IDNO:59, (ii) a p40 subunit protein comprising a fragment of a protein of(i), wherein said fragment is at least 40 amino acids in length, and(iii) a p40 subunit protein comprising at least 31 contiguous aminoacids identical in sequence to at least 31 contiguous amino acids of anamino acid sequence selected from the group consisting of SEQ ID NO:53and SEQ ID NO:59. The p35 subunit is preferably selected from the groupconsisting of (i) a p35 subunit protein having an amino acid sequencethat is at least 84 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35subunit protein comprising a fragment of a protein of (i), wherein saidfragment is at least 40 amino acids in length, and (iii) a p35 subunitprotein comprising at least 31 contiguous amino acids identical insequence to at least 31 contiguous amino acids of an amino acid sequenceselected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50.Preferred amino acid sequences have an at least 23 contiguous amino acidregion identical with a 23 contiguous amino acid region, an at least 30contiguous amino acid region identical with a 30 contiguous amino acidregion, an at least 40 contiguous amino acid region identical with a 40contiguous amino acid region, an at least 50 contiguous amino acidregion contiguous with a 50 contiguous amino acid region, an at least 75contiguous amino acid region contiguous with a 75 contiguous amino acidregion, preferably an at least 100 contiguous amino acid regioncontiguous with a 100 contiguous amino acid region, and most preferablya full-length protein identical in sequence to a full-length protein ofan amino acid sequence selected from the group consisting of SEQ IDNO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59. In anotherembodiment, preferred proteins have an amino acid sequence that is atleast 84 percent identical, at least 86 percent identical, at least 88percent identical, preferably at least 90 percent identical, and morepreferably at least about 95 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:47, SEQ ID NO:50, SEQ IDNO:53, and SEQ ID NO:59, and a fragment thereof of at least 30 aminoacids, at least 50 amino acids, at least 75 amino acids, preferably atleast 100 amino acids, and more preferably a full-length protein.

In a preferred embodiment, an IL-12 single chain protein of the presentinvention has a function selected from the group consisting of: (i)eliciting an immune response against an IL-12 protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:62 and SEQID NO:67, (ii) selectively binding to an antibody raised against anIL-12 protein having an amino acid sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36,SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQ IDNO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12 activity.

One embodiment of the present invention is a therapeutic compositionthat, when administered to a animal in an effective manner, is capableof protecting that animal from a disease such as, for example, allergy,cancer or inflammation. Therapeutic compositions of the presentinvention include protective compounds that are capable of regulatingfeline IL-18, feline caspase-1, or feline or canine IL-12 proteinamounts and/or activity. A protective compound of the present inventionis capable of regulating feline IL-18, feline caspase-1, or feline orcanine IL-12 activity and/or availability. Examples of protectivecompounds related to feline and canine proteins, of the presentinvention include an isolated antibody that selectively binds to eitherfeline IL-18, feline caspase-1, or feline or canine IL-12 or otherinhibitors or activators of feline IL-18, feline caspase-1, or feline orcanine IL-12 activity or amount. Other examples of protective compoundsinclude an isolated nucleic acid molecule of the present invention; anisolated protein of the present invention; a mimetope of a protein ofthe present invention, a multimeric form of any of said proteins, or aninhibitor identified by its ability to inhibit the activity of any ofsaid proteins; such an inhibitor can inhibit binding of the respectiveprotein with its receptor, or inhibit the activity of the respectiveprotein. Methods to perform such assays to measure binding and/oractivity of protein of the present invention are known to those of skillin the art, and are described, for example, in Janeway et al., ibid. Assuch, these protective compounds may include antibodies, peptides,substrate analogs, and other large or small molecules which can beorganic or inorganic. As used herein, a protective compound refers to acompound, that when administered to an animal in an effective manner, isable to treat, ameliorate, and/or prevent a disease due to allergy,cancer or infection. Examples of proteins, nucleic acid molecules,antibodies and/or inhibitors of the present invention are disclosedherein.

The present invention also includes a therapeutic composition comprisingat least one compound of the present invention in combination with atleast one additional therapeutic compound. Examples of such compoundsare disclosed herein.

The efficacy of a therapeutic composition of the present invention toprotect an animal from a disease mediated by feline IL-18, felinecaspase-1, or feline or canine IL-12 can be tested in a variety of waysincluding, but not limited to, detection of protective antibodies(using, for example, proteins or mimetopes of the present invention),detection of the amount of feline IL-18, feline caspase-1, or feline orcanine IL-12, or detection of cellular immunity within the treatedanimal. Therapeutic compositions can be tested in animal models such asmice. Such techniques are known to those skilled in the art.

Therapeutic compounds of the present invention can be administered toany animal susceptible to such therapy, preferably to mammals, and morepreferably to dogs, cats, humans, ferrets, horses, cattle, sheep and/orother pets, economic food animals, and/or zoo animals. Preferred animalsinclude dogs and cats.

A therapeutic composition of the present invention is administered to ananimal in an effective manner such that. the composition is capable ofregulating an immune response in that animal. Therapeutic compositionsof the present invention can be administered to animals prior to theonset of a disease (i.e. as a preventative vaccine) and/or can beadministered to animals after onset of a disease in order to treat thedisease (i.e. as a therapeutic vaccine). Preferred diseases to preventand/or treat include autoimmune diseases, allergic reactions, infectiousdiseases, tumor development, inflammatory diseases and/or graftrejection. In one embodiment, a therapeutic composition of the presentinvention is administered with an antigen to enhance an immune responseagainst that antigen. Such administration can include, but is notlimited to, oral, intravenous, intramuscular, intra ocular, mucosal,intranasal, subcutaneous, topical or transdermal application. In orderto protect an animal from disease, a therapeutic composition of thepresent invention is administered to the animal in an effective mannersuch that the composition is capable of protecting that animal from adisease. Therapeutic compositions of the present invention can beadministered to animals prior to disease in order to prevent diseaseand/or can be administered to animals after disease occurs. The exactdose, administration regimen, and administration route of therapeuticcompositions of the present invention can be determined by one skilledin the art. A suitable single dose is a dose that is capable ofregulating the immune response in an animal when administered one ormore times over a suitable time period. For example, a preferred singledose of a protein, mimetope or antibody therapeutic composition is fromabout 1 microgram (μg) to about 10 milligrams (mg) of the therapeuticcomposition per kilogram body weight of the animal. Booster vaccinationscan be administered from about 2 weeks to several years after theoriginal administration. Booster administrations preferably areadministered when the immune response of the animal becomes insufficientto protect the animal from disease. A preferred administration scheduleis one in which from about 10 μg to about 1 mg of the therapeuticcomposition per kg body weight of the animal is administered from aboutone to about two times over a time period of from about 2 weeks to about12 months.

A therapeutic composition of the present invention can include at leastone of the following: excipient, an adjuvant and a carrier. Therapeuticcompositions of the present invention can be formulated in an excipientthat the animal to be treated can tolerate. Examples of such excipientsinclude water, saline, Ringer's solution, dextrose solution, Hank'ssolution, and other aqueous physiologically balanced salt solutions.Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, ortriglycerides may also be used. Other useful formulations includesuspensions containing viscosity enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non- liquid formulation, theexcipient can comprise dextrose, human serum albumin, preservatives,etc., to which sterile water or saline can be added prior toadministration.

Therapeutic compositions of the present invention can include anadjuvant. Adjuvants are agents that are capable of enhancing the immuneresponse of an animal to a specific antigen. Suitable adjuvants include,but are not limited to, cytokines, chemokines, and compounds that inducethe production of cytokines and chemokines (e.g., granulocyte macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), macrophage colony stimulating factor (M-CSF), colonystimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2),interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5),interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8),interleukin 10 (IL-10), interferon gamma, transforming growth factorbeta, RANTES (regulated upon activation, normal T cell expressed andpresumably secreted), macrophage inflammatory proteins (e.g., MIP-1alpha and MIP-1 beta), and Leishmania elongation initiating factor(LEIF); bacterial components (e.g., endotoxins, in particularsuperantigens, exotoxins and cell wall components); aluminum-basedsalts; calcium-based salts; silica; polynucleotides; toxoids; serumproteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter'sTitermax™ adjuvant (Vaxcel™, Inc. Norcross, GA), Ribi adjuvants (RibiImmunoChem Research, Inc., Hamilton, MT); and saponins and theirderivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Proteinadjuvants of the present invention can be delivered in the form of theprotein themselves or of nucleic acid molecules encoding such proteinsusing the methods described herein.

Therapeutic compositions of the present invention can include a carrier.Carriers include compounds that increase the half-life of a protectivecompound in the treated animal. Suitable carriers include, but are notlimited to, polymeric controlled release vehicles, biodegradableimplants, liposomes, other lipid or lipid containing formulations,including cationic lipids or lipid mixtures including cationic lipids,bacteria, viruses, other cells, oils, esters, and glycols.

A therapeutic composition can be a controlled release formulation thatis capable of slowly releasing a protective compound of the presentinvention into an animal. As used herein, a controlled releaseformulation comprises a composition or protective compound of thepresent invention in a controlled release vehicle. Suitable controlledrelease vehicles include, but are not limited to, biocompatiblepolymers, other polymeric matrices, capsules, microcapsules,microparticles, bolus preparations, osmotic pumps, diffusion devices,liposomes, lipospheres, other lipids or lipid-containing formulationsand transdermal delivery systems. Other controlled release formulationsof the present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable, i.e., bioerodible.

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to regulate an immuneresponse in an animal. The therapeutic composition is preferablyreleased over a period of time ranging from about 1 to about 12 months.A controlled release formulation of the present invention is capable ofeffecting a treatment preferably for at least about 1 month, morepreferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a therapeutic protein ortherapeutic RNA (e.g. antisense RNA, ribozyme, triple helix forms or RNAdrug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e. not packaged in a viral coat or cellularmembrane) nucleic acid as a genetic vaccine (e.g. as naked DNA or RNAmolecules, such is taught, for example, in Wolff et al., 1990, Science247, p 1465-68) or (b) administering a nucleic acid molecule packaged asa recombinant virus vaccine or as a recombinant cell vaccine (i.e. thenucleic acid molecule is delivered by a viral or cellular vehicle).

One embodiment of a therapeutic composition of the present invention isa naked nucleic acid, a recombinant virus or a recombinant cell vaccineor therapy. Naked nucleic acid molecules of the present invention can beadministered by a variety of methods. Suitable delivery methods include,for example, intramuscular injection, subcutaneous injection,intradermal injection, intradermal scarification, particle bombardment,oral application, topical application and nasal application, withintramuscular injection, intradermal injection, intradermalscarification and particle bombardment being preferred. A preferredsingle dose of a naked nucleic acid molecule ranges from about 1nanogram (ng) to about 1 milligram (mg), depending on the route ofadministration and/or method of delivery, as can be determined by thoseskilled in the art. Examples of administration methods are disclosed,for example, in U.S. Pat. No. 5,204,253, by Bruner, et al., issued Apr.20, 1993, PCT Publication No. WO 95/19799, published Jul. 27, 1995, byMcCabe, and PCT Publication No. WO 95/05853, published Mar. 2, 1995, byCarson, et al. Naked nucleic acid molecules of the present invention canbe contained in an aqueous excipient (e.g., phosphate buffered saline)and/or with a carrier (e.g., lipid-based vehicles), or it can be boundto microparticles (e.g., gold particles).

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a protective protein orprotective RNA, e.g., antisense RNA, ribozyrne, triple helix form or RNAdrug, in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked, i.e., not packaged in a viral coat or cellularmembrane, nucleic acid as a genetic therapy or vaccine, e.g., as nakedDNA or RNA molecules, such as is taught, for example in Wolff et al.,1990, Science 247, 1465-1468, or (b) administering a nucleic acidmolecule packaged as a recombinant virus therapy or vaccine or as arecombinant cell therapy or vaccine, i.e., the nucleic acid molecule isdelivered by a viral or cellular vehicle.

A genetic, i.e., naked nucleic acid, therapy vaccine of the presentinvention includes a nucleic acid molecule of the present invention andpreferably includes a recombinant molecule of the present invention thatpreferably is replication or otherwise amplification, competent. Agenetic therapy or vaccine of the present invention can comprise one ormore nucleic acid molecules of the present invention operatively linkedto a transcriptional control sequence in the form of, for example, adicistronic recombinant molecule. Preferred viral vectors include thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picomaviruses, and/or retroviruses, with those based on alphaviruses(such as sindbis or Semliki forest virus) species-specific herpesvirusesand/or poxviruses being particularly preferred. Any suitabletranscription control sequence can be used, including those disclosed assuitable for protein production. Particularly preferred transcriptioncontrol sequences include cytomegalovirus immediate early (preferably inconjunction with Intron-A), Rous sarcoma virus long terminal repeat, andtissue-specific transcription control sequences, as well astranscription control sequences endogenous to viral vectors if viralvectors are used. The incorporation of a “strong” polyadenylation signalis also preferred.

Genetic therapies and vaccines of the present invention can beadministered in a variety of ways, with intramuscular, subcutaneous,intradermal, transdermal, intranasal, topical and oral routes ofadministration being preferred. A preferred single dose of a genetictherapy or vaccine ranges from about 1 nanogram (ng) to about 600 μg,depending on the route of administration and/or method of delivery, ascan be determined by those skilled in the art. Suitable delivery methodsinclude, for example, by injection, as drops, aerosolized and/ortopically. Genetic therapies or vaccines of the present invention can becontained in an aqueous excipient, e.g., phosphate buffered saline,alone or in a carrier, e.g., lipid-based vehicles. One embodiment is anucleic acid-lipid complex, preferably a nucleic acid-cationic lipidcomplex.

A recombinant virus therapy or vaccine of the present invention includesa recombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient and/or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picomaviruses, and retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses, such as Sindbis virus, raccoonpoxviruses, species-specific herpesviruses and species-specificpoxviruses. An example of methods to produce and use recombinant virustherapies and vaccines is disclosed in U.S. Pat. No. 5,766,602, Xiong etal., issued Jun. 16, 1998; U.S. Pat. No. 5,753,235, Haanes et al.,issued May 19, 1998; and U.S. Pat. No. 5,804,197, Haanes et al., issuedSep. 8, 1998, all of which are incorporated by reference herein in theirentireties.

When administered to an animal, a recombinant virus therapy or vaccineof the present invention infects cells within the immunized animal anddirects the production of a protective protein or RNA nucleic acidmolecule that is capable of protecting the animal from a disease. Forexample, a recombinant virus vaccine comprising a feline IL-18 nucleicacid molecule of the present invention can be administered according toa protocol that results in the animal producing a sufficient immuneresponse to protect itself from a disease mediated by IL-18. In anotherembodiment of the present invention a feline IL-18 nucleic acid moleculecan be used as therapy to treat a disease. A recombinant virus vaccinecomprising a feline IL-18 nucleic acid molecule can be administered toan animal with clinical signs of disease according to a protocol thatresults in reduction and/or termination of clinical signs of disease. Apreferred single dose of a recombinant virus therapy or vaccine of thepresent invention is from about 1×10⁴ to about 1×10⁸ virus plaqueforming units (pfu) per kilogram body weight of the animal.Administration protocols are similar to those described herein forprotein-based vaccines, with subcutaneous, intramuscular, intranasal,topical and oral administration routes being preferred.

A recombinant cell therapy or vaccine of the present invention includesrecombinant cells of the present invention that express at least oneprotein of the present invention. Preferred recombinant cells for thisembodiment include Salmonella, E. coli, Listeria, Mycobacterium, S.frugiperda, yeast, (including Saccharomyces cerevisiae and Pichiapastoris), BHK, CV-1, myoblast G8, COS, e.g.,COS-7, Vero, MDCK and CRFKrecombinant cells. Recombinant cell therapy or vaccines of the presentinvention can be administered in a variety of ways but have theadvantage that they can be administered orally, preferably at dosesranging from about 10⁸ to about 10¹² cells per kilogram body weight.Administration protocols are similar to those described herein forprotein-based vaccines. Recombinant cell vaccines can comprise wholecells, cells stripped of cell walls or cell lysates.

In one embodiment of the present invention, a method to regulate animmune response in an animal by administering the therapeutic compoundto an animal preferably a canine or feline, wherein the compositioncomprises a component selected from the group consisting of anexcipient, an adjuvant and a carrier.

Proteins of the present invention can be used to develop regulatorycompounds including inhibitors and activators that, when administered toan animal in an effective manner, are capable of protecting that animalfrom disease mediated by IL-18, caspase-1 or IL-12. Preferred regulatorycompounds derived from the present invention include inhibitors andactivators. In accordance with the present invention, the ability of aregulatory compound, including an inhibitor or activator, of the presentinvention to protect a felid or canid from disease mediated by IL-18,caspase-1 or IL-12 refers to the ability of that compound to, forexample, treat, ameliorate or prevent a disease mediated by IL-18,caspase-1or IL-12 in that animal.

An IL-18, caspase-1or IL-12 single chain inhibitor of the presentinvention is identified by its ability to bind to, modify, or otherwiseinteract with, an IL-18, caspase-1 or IL-12 single chain protein of thepresent invention, thereby inhibiting the activity of the protein.Suitable inhibitors of activity are compounds that inhibit the activityof the proteins of the present invention in at least one of a variety ofways: (1) by binding to or otherwise interacting with or otherwisemodifying the protein binding, (2) by interacting with other regions ofthe protein to inhibit activity, for example, by allosteric interaction,and (3) by binding to or otherwise interacting with or otherwisemodifying a protein receptor binding site such that the protein is lesslikely to bind to the protein receptor binding site. Inhibitors ofIL-18, caspase-1 and IL-12 single chain proteins are preferablyrelatively small compounds.

An embodiment of the present invention includes use of one of thefollowing methods to identify a compound capable of regulating an immuneresponse in an animal: (a) contacting an isolated feline IL-18 proteinwith a putative inhibitory compound under conditions in which, in theabsence of the compound, the protein has T cell stimulating activity;and determining if the putative inhibitory compound inhibits theactivity; (b) contacting an isolated feline caspase-1 protein with aputative inhibitory compound under conditions in which, in the absenceof the compound, the protein cleaves precursor IL-18 resulting in abiologically active mature IL-18; and determining if the putativeinhibitory compound inhibits the activity; and Ĉ) contacting an isolatedIL-12 single chain protein with a putative inhibitory compound underconditions in which, in the absence of the compound, the protein has Tcell proliferation stimulating activity; and determining if the putativeinhibitory compound inhibits the activity.

A variety of methods are known to one skilled in the art to detectbinding of an IL-18, caspase-1 or IL-12 protein to its binding partner(e.g., an antibody or receptor, as appropriate). Such methods can beused to detect IL-12, casp-1, or IL-18, or Abs or other binding partnersthereof in a biological sample or to produce inhibitors of suchinteractions. Such methods include, but are not limited to an assay inwhich, for example, IL-18 and an IL-18 binding partner can interactand/or bind to each other, using, for example, the yeast two-hybridsystem, see for example, Luban, et al. 1995, Curr. Opin. Biotechnol., 6,59-64; and identifying those proteins that specifically bind to theIL-18 protein binding domain. Additional methods to identifyprotein-protein interactions include Biacore® screening, confocalimmunofluorescent microscopy, UV cross-linking, andimmunoprecipitations. An example of a protein binding domain is anIL-18-binding domain, and a protein that would bind to an IL-18-bindingdomain would be IL-18. Additional teachings of general characteristicsof reagents for use in the detection of binding between two moieties(e.g., between IL-18 and its receptor) as well as methods to produce anduse such reagents are disclosed, for example, in U.S. Pat. No.5,958,880, issued Sep. 28, 1999, by Frank et al.; and PCT InternationalPublication No. WO 99/54349, published Oct. 28, 1999, by McCall et al.;each of these references is incorporated by reference herein in itsentirety; furthermore, the disclosed reagents and methods areincorporated by reference herein in their entireties. It is to be notedthat although the reagents and methods disclosed in each of thecitations do not relate to the proteins, nucleic acid molecules,antibodies and inhibitors of the present invention per se, the disclosedreagents and methods are applicable by those skilled in the art toreagents, kits and detection methods of the present invention.Furthermore, proteins of the present invention can be used to developregulatory compounds including inhibitors and activators that, whenadministered to a canid or felid in an effective manner are capable ofprotecting and treating that felid or canid from disease mediated byIL-18, caspase-1 or IL-12.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention. Thefollowing examples include a number of recombinant DNA and proteinchemistry techniques known to those skilled in the art; see, forexample, Sambrook et al., ibid.

EXAMPLE 1

Identification of the nucleic acid molecules of the feline IL-18 isunexpected because initial attempts to isolate feline IL-18 nucleic acidmolecules using standard cDNA screening techniques were unsuccessful.

This example describes the isolation, sequencing and expression ofnucleic acid molecules encoding feline IL-18 proteins of the presentinvention.

A. Feline IL-18 nucleic acid molecules were isolated as follows: A cDNAmitogen library was prepared from cat peripheral blood lymphocytesstimulated with ConA for 4 hours as previously described in Example 2 ofPCT Publication No. WO 99/61618, entitled “Canine and FelineImmunoregulatory Proteins, Nucleic Acid Molecules, and Uses Thereof,”inventors Gek-Kee Sim, Shumin Yang, Matthew Dreitz, and RamaniWonderling, filed May 28, 1999, which is incorporated by referenceherein in its entirety. An aliquot of this library was used as atemplate to isolate a feline IL-18 nucleic acid molecule by polymerasechain reaction (PCR). PCR amplification was performed using Amplitaq DNApolymerase™ (available from PE Applied Biosystems Inc, Foster City,Calif.). Two overlapping nucleic acid molecules encoding partial lengthfeline IL-18 proteins were obtained by using IL-18 specific primers incombination with cDNA library vector specific primers. All primers camefrom Life Technologies, Gaithersburg, MD. The sequence of the vectorforward primer (T3 primer) was 5′ GCCAAGCTCG AAATTAACCC TCACTAAAGG 3′(SEQ ID NO:72), and that of the vector reverse primer (T7 primer) was 5CGACGGCCAG TGAATTGTAA TACGACTC 3′ (SEQ ID NO:73). The sequence of theIL-18-specific forward primer (IL-18 Forward 85) was 5′ AGTGATGAAGGCCTGGAATC AGATTACTTT G 3′ (SEQ ID NO:74) and the sequence of theIL-18-specific reverse primer (IL-18 Reverse 435) was 5′ ATGGCCTGGAACACTTCTCT GAAAGAATAT GA 3′ (SEQ ID NO:75). The first PCR amplificationwas done using T3 primer and IL-18 Reverse 435 primer and the second PCRamplification was done using IL-18 Forward 85 primer and T7 primer. ThePCR profile for both reactions were as follows: one initial denaturationstep at 94° C. for 5 minutes; then 43 cycles of the following: 94° C.for 30 seconds, then 59° C. for 30 seconds, then 72° C. for 2 minutes;followed by a final extension at 72° C. for 7 minutes. The PCR productsfrom both reactions were cloned into the TA-Cloning vector (availablefrom Invitrogen, San Diego, Calif.) and the nucleic acid molecules weresequenced using an ABI Prism™ Model 377 Automatic DNA Sequencer(available from PE Applied Biosystems Inc.). DNA sequencing reactionswere performed using Prism™ dRhodamine Terminator Cycle Sequencing ReadyReaction kits (available from PE Applied Biosystems Inc.). The PCRproduct from the first PCR amplification was sequenced and found tocontain 514 nucleotides and was denoted herein as nFeIL-18₅₁₄ (5′-endpartial clone) with a coding strand of SEQ ID NO:1, and a complementarystrand of SEQ ID NO:3. The PCR product from the second PCR amplificationwas sequenced and found to contain 502 nucleotides and was denotedherein as nFeIL-18₅₀₂ (3′-end partial clone) with a coding strand of SEQID NO:4, and a complementary strand of SEQ ID NO:6. These two nucleicacid molecules shared more than 280 base pairs (bp) and togetherprovided the sequence for the complete feline IL-18 open reading frame.Translation of SEQ ID NO:1 suggests that nucleic acid moleculenFeIL-18-N₅₁₄ encodes an N-terminal portion of PFeIL-18-N protein, ofabout 133 amino acids, denoted herein as PFe IL-18-N₁₃₃, the amino acidsequence of which is presented in SEQ ID NO:2, assuming an open readingframe having an initiation codon spanning from nucleotide 114 throughnucleotide 116 of SEQ ID NO:1 and a stop codon spanning from nucleotide510 through nucleotide 512 of SEQ ID NO:1. Translation of SEQ ID NO:4suggests that nucleic acid molecule nFeIL-18-C₅₀₂ encodes an C-terminalportion of PFeIL-18-C protein, of about 154 amino acids, denoted hereinas PFe IL-18-C₁₅₄, the amino acid sequence of which is presented in SEQID NO:5, assuming an open reading frame having an initiation codonspanning from nucleotide 3 through nucleotide 5 of SEQ ID NO:4 and astop codon spanning from nucleotide 462 through nucleotide 464 of SEQ IDNO:4.

Based on the sequence data obtained from these two nucleic acidmolecules two new primers were made to isolate a cDNA encodingfull-length feline IL-18. The IL-18 Full Forward primer sequence was 5′AACTATTGAG CACAGGGATA AAGATGACTG 3′ (SEQ ID NO:76) and IL-18 FullReverse primer sequence was 5′ AATATCTAAT TCTTGTTTTG AACAGTGAAC ATT 3′(SEQ ID NO:77). The PCR amplification was performed using these twoprimers and Amplitaq DNA polymerase™ (available from PE AppliedBiosystems Inc.) and an aliquot of the cDNA library prepared from catperipheral blood lymphocytes stimulated with ConA for 4 hours. The PCRprofile was as follows: one initial denaturation step at 94° C. for 5minutes; then 43 cycles of the following: 94° C. for 30 seconds, then53° C. for 30 seconds, then 72° C. for 90 seconds; followed by a finalextension at 72° C. for 7 minutes. The PCR product was cloned into theTA-Cloning vector (available from Invitrogen, San Diego Calif.) and thenucleic acid molecule insert was sequenced using an ABI PRISM™ Model 377Automatic DNA Sequencer (available from PE Applied Biosystems Inc.). DNAsequencing reactions were performed using PRISM™ drhodamine TerminatorCycle Sequencing Ready Reaction kits (available from PE AppliedBiosystems Inc.). This PCR product the FeIL-18 containing region ofwhich is denoted nFeIL-18₆₀₇ was found to encode a full-length FeIL-18protein. The nucleotide sequence of the coding strand of nFeIL-18₆₀₇ isrepresented herein as SEQ ID NO:7, and its complement is denoted by SEQID NO:10. Translation of the open reading frame in SEQ ID NO:7, denotedherein as nFeIL-18₅₇₆, the coding strand of which is denoted SEQ IDNO:9, and the complementary strand denoted SEQ ID NO:41 suggests thatfeline IL-18 encodes a protein containing 192 amino acids, referred toherein as PFeIL-18₁₉₂, with a SEQ ID NO:8. The nucleic acid sequenceencoding feline IL-18 protein assumes an open reading frame in which thefirst codon spans from nucleotide 24 through 26 of SEQ ID NO:7, and thelast codon spans from nucleotide 597 through nucleotide 599 of SEQ IDNO:7. The encoded protein has a predicted molecular weight of about 21.3kiloDaltons (kDa) for the precursor protein. The IL-18 precursor proteindoes not contain a signal sequence; in order for IL-18 to bebiologically active the precursor is cleaved by caspase-1. The putativecaspase-1 cleavage site is between amino acid positions 35 and 36 of thefeline IL-18 precursor protein. Nucleic acid molecule nFeIL-18₄₇₁, whichencodes the mature protein contains a coding strand with SEQ ID NO:11,and a complementary strand with SEQ ID NO:13. The amino acid sequence ofthe mature protein, denoted herein as PFeIL-18₁₅₇is SEQ ID NO:12 and themature protein has a predicted molecular weight of about 17.4 kDa.Sequence analysis was performed using DNAsis™, available from HitachiSoftware, San Bruno, Calif. using the alignment settings of: gap penaltyset at 5, k-tuple set at 3, number of top diagonals set at 5, windowsize set at 5, fixed gap penalty set at 10 and floating gap penalty setat 10.

B. In an attempt to express a mature feline IL-18 protein in a mammaliancell line, the region encoding only the mature IL-18 protein (SEQ IDNO:11) was isolated from the feline cDNA library described in Example 1Ausing the following primers: IL-18 MatNgo Forward primer which has thesequence 5′ TATGCCGGCT ACTTTGGCAA GCTTGAACAT AAACTC 3′ (SEQ ID NO:78)and IL-18 MatXho Reverse primer which has the sequence 5′ GGCCTCGAGCTAATTCTTGT TTTGAACAGT GAACATT 3′ (SEQ ID NO:79). The PCR amplificationwas performed using these two primers and Amplitaq DNA polymerase™(available from PE Applied Biosystems Inc.) and an aliquot of the cDNAlibrary prepared from cat peripheral blood lymphocytes stimulated withConA for 4 hours. The PCR profile was as follows: one initialdenaturation step at 94° C. for 5 minutes; then 43 cycles of thefollowing: 94° C. for 30 seconds, then 53° C. for 30 seconds, then 72°C. for 90 seconds; followed by a final extension at 72° C. for 7minutes. The PCR products were digested with Ngo MI and Xho Irestriction enzymes (available from New England Biolabs, Beverly, Mass.)and ligated downstream of nucleotides encoding a tissue plasminogenactivator (tPA) signal sequence contained in the CMV-IntronA-tPA vector(available from Invitrogen). The construct was sequenced using an ABIPrism™ Model 377 Automatic DNA Sequencer (available from PE AppliedBiosystems Inc.). DNA sequencing reactions were performed using Prism™dRhodamine Terminator Cycle Sequencing Ready Reaction kits (availablefrom PE Applied Biosystems Inc.). This construct encoded the maturefeline IL-18 protein with the tPA signal sequence. When Chinese hamsterovary (CHO) cells (available from ATCC, Rockville, Md.) were transientlytransfected with this construct, using techniques known to those skilledin the art and cell pellets and supernatants were harvested after 48hrs. Western analysis was performed on the cell pellets and supernatantsamples using a polyclonal antibody against human IL-18 (available fromBiosource International, Camarillo, Calif.). A faint band of theexpected size (about 17.4 kDa) was detected in the cell pellet and notin the supernatant, indicating that IL-18 is produced by this constructbut it is not exported out of the cell at detectable levels. While notbeing bound by theory, it is believed that caspase-1 plays a key role inthe processing of native IL-18 precursor in cells where IL-18 isproduced, co-expression of full-length feline IL-18 along with thefeline caspase-1 may be necessary for the proper processing of the IL-18precursor and enhanced secretion of the processed IL-18 maturepolypeptide.

EXAMPLE 2

This example describes the isolation and sequencing of nucleic acidmolecules encoding feline caspase-1 proteins of the present invention.

Feline caspase-1 nucleic acid molecules were isolated as follows: A cDNAmitogen library was prepared from cat peripheral blood lymphocytesstimulated with ConA for 4 hours as described in Example 1. An aliquotof this library was used as a template to isolate a feline caspase-1 bypolymerase chain reaction (PCR). PCR amplification was performed usingAmplitaq DNA polymerase™ (available from PE Applied Biosystems Inc.).The forward and reverse primers were designed based on human caspase-1sequences. The forward primer (Casp-1 For) had a sequence of 5′ATGGCCGACA AGGTCCTGAA GGAGAAGA 3′ (SEQ ID NO:80) and the reverse primer(Casp-1 Rev) had a sequence of 5′ TTAATGTCCT GGGAAGAGGT AGAAACATCT TGT3′ (SEQ ID NO:81). The PCR profile was as follows: one initialdenaturation step at 94° C. for 5 minutes; then 43 cycles of thefollowing: 94° C. for 45 seconds, then 53° C. for 45 seconds, then 72°C. for 2 minutes; followed by a final extension at 72° C. for 7 minutes.The PCR product was cloned into the TA-Cloning vector (available fromInvitrogen, San Diego Calif.) and sequenced using an ABI Prism™ Model377 Automatic DNA Sequencer (available from PE Applied Biosystems Inc.).DNA sequencing reactions were performed using Prism™ dRhodamineTerminator Cycle Sequencing Ready Reaction kits (available from PEApplied Biosystems Inc.). The PCR product was found to contain thecomplete full-length feline caspase-1 except for the primer region whichwas based on the human caspase-1 sequence. The nucleotide sequence ofthe coding strand of this PCR product is represented herein asnFeCasp-1₁₂₃₃ with a SEQ ID NO:14, and its complement is denoted by SEQID NO:16. Translation of SEQ ID NO:14 suggests that nucleic acidmolecule nFeCasp-1₁₂₃₃ encodes a full-length nFeCasp-1₁₂₃₃ protein, ofabout 410 amino acids, denoted herein as PFeCasp-1₄₁₀, the amino acidsequence of which is presented in SEQ ID NO:15, assuming an open readingframe having an initiation codon spanning from nucleotide 1 throughnucleotide 3 of SEQ ID NO:14 and a stop codon spanning from nucleotide408 through nucleotide 410 of SEQ ID NO:14.

Additional primers were made based on the feline caspase-1 sequence ofnFeCasp-1₁₂₃₃ in order to obtain two nucleic acid molecules spanning the5′ and 3′ end of the feline caspase-1 open reading frame. Two felinecaspase-1 nucleic acid molecules were generated using feline caspase-1specific primers in combination with cDNA library vector specificprimers. The sequence of the vector forward primer (T3 primer) was 5′GCCAAGCTCG AAATTAACCC TCACTAAAGG 3′ (SEQ ID NO:72), and that of thevector reverse primer (T7 primer) was 5′ CGACGGCCAG TGAATTGTAA TACGACTC3′ (SEQ ID NO:73). The sequence of the feline caspase-1-specific forwardprimer (Casp 271 Forward) was 5′ TCAAGCCCAC AATCTGGAAA TTCTCA 3′ (SEQ IDNO:82) and the sequence of the feline caspase-1 -specific reverse primer(Casp 895 Reverse) was 5′ CTGGAGAGTC ACTGATCAAC AGTTCC 3′ (SEQ IDNO:83). The first PCR amplification was done using T3 primer and Casp895 Reverse primer and the second PCR amplification was done using Casp271 Forward primer and T7 primer. The PCR profile for both reactions wasas follows: one initial denaturation step at 94° C. for 5 minutes; then43 cycles of the following: 94° C. for 45 seconds, then 52° C. for 45seconds, then 72° C. for 2 minutes; followed by a final extension at 72°C. for 7 minutes. The PCR products from both reactions that were greaterthan or equal to 1 kb were gel purified and cloned into the TA-Cloningvector (available from Invitrogen) and the nucleic acid molecules weresequenced using an ABI Prism™ Model 377 Automatic DNA Sequencer(available from PE Applied Biosystems Inc.). DNA sequencing reactionswere performed using Prism™ dRhodamine Terminator Cycle Sequencing ReadyReaction kits (available from PE Applied Biosystems Inc.). The nucleicacid molecules obtained from these two PCR products represented twonucleic acid molecules of feline caspase-1. The region of the first PCRamplification was sequenced and found to contain 527 nucleotides denotedherein as nFeCasp-1-N₅₂₇ (5′-end partial clone) with a coding strand ofSEQ ID NO:17, and a complementary strand of SEQ ID NO:19. The region ofthe second PCR amplification was sequenced and found to contain 500nucleotides denoted here as nFeCasp-1-C₅₀₀ (3′-end partial clone) with acoding strand of SEQ ID NO:20, and a complementary strand of SEQ IDNO:22. Translation of SEQ ID NO:17 suggests that nucleic acid moleculenFeCasp-1-N₅₂₆ encodes an N-terminal portion of PFeCasp-1-N protein, ofabout 169 amino acids, denoted herein as PFeCasp-1-N₁₆₉, the amino acidsequence of which is presented in SEQ ID NO:18, assuming an open readingframe having an initiation codon spanning from nucleotide 18 throughnucleotide 20 of SEQ ID NO:17 and a stop codon spanning from nucleotide522 through nucleotide 524 of SEQ ID NO:17. Translation of SEQ ID NO:20suggests that nucleic acid molecule nFeCasp-1-C₅₀₀ encodes an C-terminalportion of PFeCasp-1-C protein, of about 120 amino acids, denoted hereinas PFeCasp-1-C₁₂₀, the amino acid sequence of which is presented in SEQID NO:2 1, assuming an open reading frame having an initiation codonspanning from nucleotide 3 through nucleotide 5 of SEQ ID NO:20 and astop codon spanning from nucleotide 360 through nucleotide 362 of SEQ IDNO:20

Based on the sequence data obtained from nucleic acid moleculesnFeCasp-1-N₅₂₇ and nFeCasp-1-C₅₀₀, two new primers were made to isolatea cDNA encoding full-length feline caspase-1. The feline caspase-1full-length forward primer (CaspBamKozFor) sequence was 5′ ACAAGGATCCACCATGGCCG ACAAGGATCT GAAGGG 3′ (SEQ ID NO:84) and feline caspase-1full-length reverse primer (CaspXbaRev) sequence was 5′ CGCCTCTAGACCTCAATTGC CAGGGAAGAG ATAGAAGTA 3′ (SEQ ID NO:85). The PCR amplificationwas performed using these two primers and Amplitaq DNA polymerase™(available from PE Applied Biosystems Inc.) and an aliquot of the cDNAmitogen library prepared from cat peripheral blood lymphocytesstimulated with ConA for 4 hours. The PCR profile was as follows: oneinitial denaturation step at 94° C. for 5 minutes; then 43 cycles of thefollowing: 94° C. for 45 seconds, then 52° C. for 45 seconds, then 72°C. for 2 minutes; followed by a final extension at 72° C. for 7 minutes.The PCR product was cloned into the TA-Cloning vector (available fromInvitrogen) and the nucleic acid molecule inserts were sequenced usingan ABI Prism™ Model 377. Automatic DNA Sequencer (available from PEApplied Biosystems Inc.). DNA sequencing reactions were performed usingPrismT™ dRhodamine Terminator Cycle Sequencing Ready Reaction kits(available from PE Applied Biosystems Inc.). This PCR product theFeCaspase-1 containing region of which is denoted nFeCasp-1₁₂₃₀ wasfound to encode the a full-length feline caspase-1 protein. Thenucleotide sequence of the coding strand of nFeCasp-1₁₂₃₀ is representedherein as SEQ ID NO 23, and its complement is denoted by SEQ ID NO:25.Translation of the open reading frame in SEQ ID NO:23, denoted here asnFeCasp-1₁₂₃₀, the coding strand of which is denoted SEQ ID NO:25suggests that feline caspase-l encodes a protein containing 410 aminoacids, referred to herein as PFeCasp-1₄₁₀, with a SEQ ID NO:24. Thenucleic acid sequence encoding the protein assumes an open reading framein which the first codon spans from nucleotide 1 through 3 of SEQ IDNO:23, and the last codon spans from nucleotide 1228 nucleotide 1230 ofSEQ ID NO:23. The encoded protein has a predicted molecular weight ofabout 45.5 kDa. The feline caspase-1protein is 9 amino acids longer thanmouse and rat caspase-1 proteins, 6 amino acids longer than dog andhuman caspase-1proteins, and 5 amino acids longer than horse caspase-1proteins. Sequence analysis was performed using DNAsis™, available fromHitachi Software, San Bruno, Cailf. using the alignment settings of: gappenalty set at 5, k-tuple set at 3, number of top diagonals set at 5,window size set at 5, fixed gap penalty set at 10 and floating gappenalty set at 10.

EXAMPLE 3

This example describes the isolation and sequencing of nucleic acidmolecules encoding feline IL-12 single chain proteins of the presentinvention.

A. A pBluescript-Linker plasmid was constructed as follows: Twocomplementary oligonucleotides, 60 nucleotides in length weresynthesized. The oligonucleotides were allowed to hybridize to eachother in solution producing a double stranded DNA fragment that wouldserve as a linker between the cDNAs encoding the p40 and p35 subunits offeline IL-12. The sequence of the sense linker was 5′ CTGCAGTGGTGGCGGTGGCG GCGGATCTAG AAACTTGCCA ACCCCTACTC CATCCCCGGG 3′ (SEQ ID NO:83)and the sequence of the antisense linker was 5′ CCCGGGGATG GAGTAGGGGTTGGCAAGTTT CTAGATCCGC CGCCACCGCC ACCACTGCAG 3′ (SEQ ID NO:84). Equimolaramounts of sense linker and antisense linker were mixed and heated to95° C. for 10 minutes in a heat block. The heat block containing thesamples was removed from the heat source and allowed to cool to roomtemperature slowly, over a period of 4 hours. Then the hybridizedoligonucleotides were digested with PstI and SmaI restriction enzymes(available from New England Biolabs, Beverly, Mass.) and ligated intopBluescript SK⁺ vector (available from Stratagene, La Jolla, Calif.)digested with the same restriction enzymes to produce pBluescript-Linkerplasmid. The presence of the linker in the ligated pBluescript-Linkerplasmid was confirmed by sequencing conducted as described in Example 1.The pBluescript-Linker plasmid contained DNA coding for the followingelements: (1) the last two C-terminal amino acid residues of the p40subunit (i.e. C,S); (2) the seven amino acid residues of the linker(i.e. GGGGGGS) (SEQ ID NO:110); and (3) the first ten N-terminal aminoacid residues of the mature p35 subunit mature protein (i.e. RNLPTPTPSP)(SEQ ID NO:111).

B. Feline IL-12 p40 nucleic acid molecule subunit was isolated asfollows: A cDNA mitogen library was prepared from cat peripheral bloodlymphocytes stimulated with ConA for 4 hours as previously described inExample 1. An aliquot of this library was used as a template to isolatea feline IL-12 p40 nucleic acid molecule subunit by polymerase chainreaction (PCR). The PCR amplification was performed using Amplitaq DNApolymerasem™ (available from PE Applied Biosystems Inc.). The sequenceof the forward primer was 5′ ATGCATCCTC AGCAGTTGGT CATCGCCT 3′ (SEQ IDNO:85), and that of the reverse primer was 5′ TGCAGGACAC GGATGCCCAGTTGCT 3′ (SEQ ID NO:86). The PCR profile was as follows: one initialdenaturation step at 94° C. for 5 minutes; then 43 cycles of thefollowing: 94° C. for 45 seconds, then 50° C. for 45 seconds, then 72°C. for 2 minutes; followed by a final extension at 72° C. for 7 minutes.PCR products were cloned into the TA-Cloning vector (available fromInvitrogen) and the nucleic acid molecule inserts were sequenced asdescribed in Example 1. One of the sequenced PCR products contained 985nucleotides and was denoted herein as nFeIL-12 p40-N₉₈₅ with a codingstrand of SEQ ID NO:55, and a complementary strand of SEQ ID NO:57.Translation of SEQ ID NO:55 suggests that nucleic acid moleculenFeIL-12p40-N₉₈, encodes an N-terminal portion of PFeIL-12p40-N protein,of about 328 amino acids, denoted herein as PFe IL-12p40-N₃₂₈, the aminoacid sequence of which is presented in SEQ ID NO:56, assuming an openreading frame having an initiation codon spanning from nucleotide 1through nucleotide 3 of SEQ ID NO:55 and a stop codon spanning fromnucleotide 982 through nucleotide 984 of SEQ ID NO:55.

This nucleic acid molecule was used as a template for a subsequent PCRreaction to obtain a full-length nucleic acid molecule. The PCRamplification was performed using Amplitaq DNA polymerase™ (PE AppliedBiosystems Inc, Foster City, Calif.). The sequence of the forward primerwas 5′ ACAGGTACCA TGCATCCTCA GCAGTTGGTC ATCGCCT 3′ (SEQ ID NO:87), andthat of the reverse primer was 5′ CTAACTGCAG GACACGGATG CCCAG 3′ (SEQ IDNO:88). The PCR profile was as follows: one initial denaturation step at94° C. for 5 minutes; then 35 cycles of the following: 94° C. for 30seconds, then 50° C. for 30 seconds, then 72° C. for 90 seconds;followed by a final extension at 72° C. for 7 minutes. This PCR product,the Fe IL-12p40 single chain subunit containing region of which isdenoted nFeIL-12 p40₉₈₇ was found to encode a full-length feline IL-12p40 single chain subunit protein. The nucleotide sequence of the codingstrand of nFeIL-12 p40₉₈₇ is represented herein as SEQ ID NO:29, and itscomplementary strand is denoted by SEQ ID NO:3 1. Translation of SEQ IDNO:29 suggests that nucleic acid molecule nFeIL-12p40₉₈₇ encodes afull-length PFeIL-12p40 protein of about 329 amino acids, denoted hereinas PFe IL-12p40₃₂₉, the amino acid sequence of which is presented in SEQID NO:30, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 of SEQ ID NO:29 and astop codon spanning from nucleotide 985 through nucleotide 987 of SEQ IDNO:29. This PCR product was digested with Kpn I and Pst I restrictionenzymes (available from New England Biolabs) and cloned into thepBluescript-Linker plasmid described in Example 3A. The resultantrecombinant molecule is referred to as fep40-linker plasmid. There is aputative cleavage site on SEQ ID NO:30, yielding the coding region for amature (i.e. lacking a signal or leader sequence) nFeIL-12p40₉₂₁ ,denoted herein as SEQ ID NO:26, with the complement denoted SEQ IDNO:28. Translation of SEQ ID NO:26 yields a mature IL-12 p40 proteindenoted PFeIL-12p40₃₀₇, also denoted herein as SEQ ID NO:27.

C. A Feline IL-12 p35 nucleic acid molecule subunit was isolated asfollows: A cDNA mitogen library was prepared from cat peripheral bloodlymphocytes stimulated with ConA for 4 hours as previously described inExample 1. An aliquot of this library was used as a template to isolatefeline IL-12 p35 subunit by polymerase chain reaction (PCR). The PCRamplification was performed using Amplitaq DNA polymerase™ (PE AppliedBiosystems Inc, Foster City, Calif.). The sequence of the forward primerwas 5′ ATGTGCCCGC CGCGTGGCC 3′ (SEQ ID NO:89), and that of the reverseprimer was 5′ CTAGGAAGCA TTCAGATAGC TCATCAT 3′ (SEQ ID NO:90). The PCRprofile was as follows: one initial denaturation step at 94° C. for 5minutes; then 43 cycles of the following: 94° C. for 45 seconds, then50° C. for 45 seconds, then 72° C. for 2 minutes; followed by a finalextension at 72° C. for 7 minutes. PCR products were cloned into theTA-Cloning vector (available from Invitrogen) and the nucleic acidmolecules were sequenced as described in Example 1. One of the sequencedPCR products contained 666 nucleotides and was denoted herein asnFeIL-12-p35₆₆₆ with a coding strand of SEQ ID NO:32, and acomplementary strand of SEQ ID NO:34. Translation of SEQ ID NO:32suggests that nucleic acid molecule nFeIL-12p35₆₆₆ encodes a full-lengthPFeIL-12p35 protein of about 222 amino acids, denoted herein as PFeIL-12p35₂₂₂, the amino acid sequence of which is presented in SEQ IDNO:33, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 of SEQ ID NO:32 and astop codon spanning from nucleotide 664 through nucleotide 666 of SEQ IDNO:32. There is a putative cleavage site on SEQ ID NO:33, yielding thecoding region for a mature (i.e. lacking a signal or leader sequence)nFeIL-12p35₅₉₁, denoted herein as SEQ ID NO:35, with the complementdenoted SEQ ID NO:37. Translation of SEQ ID NO:26 yields a mature IL-12p35 protein denoted PFeIL-12p35₁₉₇, also denoted herein as SEQ ID NO:36.SEQ ID NO:26 was digested with Sma I and Not I restriction enzymes(available from New England Biolabs) and cloned into the fep40-linkerplasmid described in Example 3B digested with the same enzymes. Theresultant recombinant molecule is referred to as fep40-linker-p35matureplasmid.

D. The fep40-linker-p35mature plasmid contained a nucleic acid moleculeencoding a feline IL-12 single chain protein of the present inventioninserted into the Kpn I and Not I sites of the pBluescript backbone.Nucleic acid molecule nFeIL-12₁₅₉₉ was sequenced as described inExample 1. The nucleotide sequence of the coding strand of nFeIL-12₁₅₉₉is represented herein as SEQ ID NO:38, and that of the complementarystrand is SEQ ID NO:40. Translation of the open reading frame in SEQ IDNO:38, suggests that nFeIL-12₁₅₉₉ encodes a protein containing 533 aminoacids, referred to herein as pFeIL-12₅₃₃, with an amino acid sequencedenoted by SEQ ID NO:39. The nucleic acid sequence encoding the proteinassumes an open reading frame in which the first codon spans fromnucleotide 1 through 3 of SEQ ID NO:38 and the last codon spans fromnucleotide 1597 nucleotide 1599 of SEQ ID NO:38. The encoded protein hasa predicted molecular weight of about 59.2 kDa. The putative signalpeptide cleavage site is between amino acid positions 22 and 23 of thep40 subunit. Nucleic acid molecule nFeIL-12₁₅₃₃, which encodes themature protein contains a coding strand with SEQ ID NO:43, and acomplementary strand with SEQ ID NO:45. The amino-acid sequence of themature protein, denoted herein as PFeIL-12₅₁₁ is SEQ ID NO:44 and themature protein has a predicted molecular weight of about 56.8 kDa.

Chinese hamster ovary (CHO) cells (available from ATCC, Rockville, Md.)were transiently transfected with fep40-linker-p35mature plasmid(containing SEQ ID NO:38) using techniques known to those skilled in theart, cell pellets and supernatants were harvested after 48 hrs. Westernanalysis was performed on the cell pellets and supernatant samples usinga polyclonal antibody against human IL-12 (available from BiosourceInternational, Camarillo, Calif.). A faint band of the expected size(about 59.2 kDa) was detected in the cell pellet and in the supernatant,indicating that IL-12 is produced by this construct at detectablelevels.

EXAMPLE 4

This example describes the isolation and sequencing of nucleic acidmolecules encoding canine IL-12 single chain proteins of the presentinvention.

A. A canine IL-12 p35 nucleic acid molecule subunit was isolated asfollows: A cDNA mitogen library was prepared from canine peripheralblood lymphocytes (PBLs) stimulated with ConA for 4 hours as describedin Example 1. Recombinant phage containing DNA encoding the p35 subunitwere identified by nucleic acid hybridization using a p³² radiolabeledprobe. The p35 probe (nCaIL-12p35TA) was generated by PCR of total RNA,prepared from ConA-stimulated PBLs in the following manner. The sequenceof the forward primer was 5′ CCATCCTGGT CCTGCTAAG C 3′ (SEQ ID NO:93)and the sequence of the reverse primer was 5′ CCATCTGGTA CATCTTCAAG TC3′ (SEQ ID NO:94). PCR amplification was performed using Amplitaq DNApolymerase™ (available from PE Applied Biosystems Inc.) using thefollowing profile: 95° C. for 2 minutes; then 30 cycles of 95° C. for 1minute, 55° C. for 1 minute, and 72° C. for 1 minute; and a finalextension at 72° C. for 10 minutes. The amplified DNA fragment waspurified with Qiagen gel purification kit, available from Qiagen, LaJolla, Cailf.) and PCR products were cloned into the TA cloning vector(available from Invitrogen Corporation, Carlsbad, Cailf.), and theresulting clones were sequenced using an ABI Prism™ Model 377 AutomaticDNA Sequencer (available from Perkin-Elmer Applied Biosystems Inc.,Foster City, Cailf.). DNA sequencing reactions were performed usingPrism™ dRhodamine Terminator Cycle Sequencing Ready Reaction kits(available from PE Applied Biosystems Inc.). Phage DNA was permanentlycross-linked to the nitrocellulose sheets using a Stratalinker®) UVcrosslinker (available from Stratagene). The plaque lifts werepre-hybridized in a solution of 6×SSC (20×SSC is 3.0 M NaCl and 0.3 Msodium citrate), 5× Denhardt's solution (50×SSC is 0.01 grams/milliliterFicoll, type 400; 0.01g/ml polyvinylpyrrolidone; and 0.01g/ml bovineserum albumin, fraction V, all available from Sigma, St. Louis, Mo.),0.5% sodium dodecyl sulfate (SDS), and 100 micrograms/ml denaturedsalmon sperm for 2 hours at 68° C. Denatured, radiolabeled probe wasadded to the pre-hybridization solution at a concentration of 1×10⁶cpm/ml and the hybridization continued for 18-24 hours at 68° C.Nonspecifically bound and unbound probe was removed by washing two timesin 2×SSC with 0.1% SDS, 30 minutes each at 68° C. and one time in 1×SSCwith 0.1% SDS, 60 minutes at 68° C. The hybridized plaque lifts wereexposed to Kodak x-ray film for approximately 18 hours. Positive phagewere plaque purified three times using the following hybridizationprotocol: phage plaques grown in solid top agar were lifted onto purenitrocellulose sheets (available from Schleicher & Schuell, Keene, NH)then denatured and neutralized by soaking the sheets in 0.5 N NaOH/1.5 MNaCl, followed by 0.5 M Tris-HCl pH7.4/1.5 M NaCl. pBluescript plasmid,containing a cDNA encoding the full-length canine IL-12 p35 subunit, wasexcised from plaque purified phage using the ExAssist™ helper phage(available from Stratagene) following the manufacturers' instructions.The nucleotide sequence of that cDNA, denoted herein as nCa IL-12p35₁₄₅₅ was verified by sequencing as described in Example 1. Thenucleic acid sequence of the coding strand of nCaIL-12p35₁₄₅₅represented as SEQ ID NO:104, and its complementary strand is SEQ IDNO:106. Translation of SEQ ID NO:104 suggests that nucleic acid moleculenCaIL-12p35₁₄₅₅ encodes an N-terminal portion of PCaIL-12p35 protein, ofabout 222 amino acids, denoted herein as PCa IL-12p35₂₂₂, the amino acidsequence of which is presented in SEQ ID NO:105, assuming an openreading frame having an initiation codon spanning from nucleotide 232through nucleotide 234 of SEQ ID NO:104 and a stop codon spanning fromnucleotide 895 through nucleotide 897 of SEQ ID NO:104.

Nucleic acid molecule nCaIL-12p35₁₄₅₅ was used as the template in PCR toobtain the coding region of the full-length form of canine IL-12 p35subunit. The sequence of the forward primer was 5′ AAAAAACCCG GGTATGTTCCAATGTTTCAA CCACTCCC 3′ (SEQ ID NO:95) and the sequence of the reverseprimer was 5′ GCGGCCGCTC GAGTTAGGAA GAGTTCAAGT AGGACATCAT TCTATTGATG G3′ (SEQ ID NO:96). PCR was performed using Pfu DNA polymerase (availablefrom Stratagene) as follows: 95° C. for 45 seconds; then 25 cycles of95° C. for 45 seconds, 55° C. for 45 seconds, 72° C. for 1 minute;followed by a final extension at 72° C. for 10 minutes. The PCR productcontains the nucleic acid sequence of canine IL-12 p35 subunit whichencodes a full-length canine IL-12 p35 subunit protein. The nucleotidesequence of the coding strand of nCaIL-12p35₆₆₆ is represented herein asSEQ ID NO:46 and its complementary strand is denoted SEQ ID NO:48.Translation of SEQ ID NO:46 suggests that nucleic acid moleculenCaIL-12p35₆₆₆ encodes a mature PCaIL-12p35 single chain protein ofabout 222 amino acids, denoted herein as PCaIL-12p35₂₂₂, the amino acidsequence of which is presented in SEQ ID NO:47, assuming an open readingframe having an initiation codon spanning from nucleotide 1 throughnucleotide 3 of SEQ ID NO:46 and a stop codon spanning from nucleotide589 through nucleotide 591 of SEQ ID NO:46. The coding sequence for themature polypeptide is encoded by SEQ ID NO:46, the coding region for amature (i.e. lacking a signal or leader sequence) nCaIL-12p35₅₉₁,denoted herein as SEQ ID NO:49, with the complement denoted SEQ IDNO:51. Translation of SEQ ID NO:49 yields a mature IL-12 p35 proteindenoted PCaIL-12p35₁₉₇, also denoted herein as SEQ ID NO:50.nCaIL-12p35₅₉₁ was digested with SmaI and XhoI restriction endonucleases(available from New England Biolabs) and ligated into thepBluescript-Linker plasmid described in Example 3A digested with thesame enzymes. The resultant recombinant molecule is referred to ascalinker-p35mature plasmid.

B. Canine IL-12 p40 nucleic acid molecule subunit was isolated asfollows: A cDNA mitogen library was prepared from canine peripheralblood lymphocytes stimulated with ConA for 4 hours as described inExample 1. Recombinant phage containing DNA encoding the p40 subunitwere identified by nucleic acid hybridization using a p³² radiolabeledprobe. The p40 probe (nCaIL-12p4OTA) was generated by PCR of total RNA,prepared from ConA stimulated PBLs in the following manner. The sequenceof the forward primer was 5′ CTTAAAGGAA CAGAAAGAAT CC 3′ (SEQ ID NO:97)and the sequence of the reverse primer was 5′ GGTATTCCCA GCTGACCTC 3′(SEQ ID NO:98). PCR amplification was performed using Amplitaq DNApolymerase™ (available from PE Applied Biosystems Inc.) using thefollowing profile: 95° C. for 2 minutes; then 30 cycles of 95° C. for 1minute, 55° C. for 1 minute, and 72° C. for 1 minute; and a finalextension at 72° C. for 10 minutes. The amplified DNA fragment waspurified with Qiagen gel purification kit, available from Qiagen, LaJolla, Cailf.) and PCR products were cloned into the TA cloning vector(available from Invitrogen Corporation, Carlsbad, Cailf.), and theresulting clones were sequenced using an ABI Prism™ Model 377 AutomaticDNA Sequencer (available from Perkin-Elmer Applied Biosystems Inc.,Foster City, Cailf.). DNA sequencing reactions were performed usingPrism™ dRhodamine Terminator Cycle Sequencing Ready Reaction kits(available from PE Applied Biosystems Inc.). Phage DNA was permanentlycross-linked to the nitrocellulose sheets using a Stratalinker®⁾ UVcrosslinker (available from Stratagene). The plaque lifts werepre-hybridized in a solution of 6×SSC (20×SSC is 3.0 M NaCl and 0.3 Msodium citrate), 5× Denhardt's solution (50×SSC is 0.01 grams/milliliterFicoll, type 400; 0.0 g/ml polyvinylpyrrolidone; and 0.0 g/ml bovineserum albumin, fraction V, all available from Sigma, St. Louis, Mo.),0.5% sodium dodecyl sulfate (SDS), and 100 micrograms/ml denaturedsalmon sperm for 2 hours at 68° C. Denatured, radiolabeled probe wasadded to the pre-hybridization solution at a concentration of 1×10⁶cpm/ml and the hybridization continued for 18-24 hours at 68° C.Nonspecifically bound and unbound probe was removed by washing two timesin 2×SSC with 0.1% SDS, 30 minutes each at 68° C. and one time in 1×SSCwith 0.1 % SDS, 60 minutes at 68° C. The hybridized plaque lifts wereexposed to Kodak x-ray film for approximately 18 hours. Positive phagewere plaque purified three times using the following hybridizationprotocol: phage plaques grown in solid top agar were lifted onto purenitrocellulose sheets (available from Schleicher & Schuell, Keene, N.H.)then denatured and neutralized by soaking the sheets in 0.5 N NaOH /1.5M NaCl, followed by 0.5 M Tris-HCl pH7.4/1.5 M NaCl. pBluescriptplasmid, containing a cDNA encoding the full-length canine IL-12 p40subunit, was excised from plaque purified phage using the ExAssist™helper phage (available from Stratagene) following the manufacturers'instructions. The nucleotide sequence of that cDNA, denoted herein asnCaIL-12p40₂₂₆₇ was verified by sequencing as described in Example 1.The nucleic acid sequence of the coding strand of nCaIL-12p40₂₂₆₇represented as SEQ ID NO:107, and its complementary strand is SEQ IDNO:109. Translation of SEQ ID NO:107 suggests that nucleic acid moleculenCaIL-12p40₂₂₆₇ encodes an PCaIL-12p40 protein, of about 329 aminoacids, denoted herein as PCa IL-12p40₃₂₉, the amino acid sequence ofwhich is presented in SEQ ID NO:108, assuming an open reading framehaving an initiation codon spanning from nucleotide 154 throughnucleotide 156 of SEQ ID NO:107 and a stop codon spanning fromnucleotide 1138 through nucleotide 1140 of SEQ ID NO:107. Full lengthcanine IL-12 p40 nucleic acid molecule was isolated as follows: Aplasmid containing full-length canine IL-12 p40 nucleic acid moleculesubunit (pCaIL-12p40) was used as a template to sub-clone canine IL-12p40 subunit by polymerase chain reaction (PCR). PCR amplification wasperformed using Amplitaq DNA polymerase™ (available from PE AppliedBiosystems Inc.). The sequence of the forward primer (Dog p40 KpnFor)was 5′ CATAGGTACC ATGCACCCTC AGCAGTTGGT CATCTCC 3′ (SEQ ID NO:99), andthat of the reverse primer (Dog p40 NsiRev) was 5′ ATCTAAATGC ATGACACAGATGCCCAGTC 3′ (SEQ ID NO:100). The PCR profile was as follows: oneinitial denaturation step at 94° C. for 5 minutes; then 35 cycles of thefollowing: 94° C. for 30 seconds, then 55° C. for 30 seconds, then 72°C. for 2 minutes; followed by a final extension at 72° C. for 7 minutes.The PCR product contains the nucleic acid sequence of canine IL-12 p40subunit along with its native signal sequence which encodes a caninefull-length IL-12 p40 subunit protein. The nucleotide sequence of thecoding strand of nCaIL-12 p40₉₈₇ is represented herein as SEQ ID NO:58and its complementary strand is denoted SEQ ID NO:60. Translation of theopen reading frame in SEQ ID NO:58, suggests that canine IL-12 p40subunit encodes a protein containing 329 amino acids, referred to hereinas PCaIL-12 p40₃₂₉ with an amino acid sequence denoted by SEQ ID NO:59.The resulting recombinant molecule is referred to as cap40-linkerplasmid. The cap40-linker plasmid was digested with Kpn I and Pst Irestriction enzymes (available from New England Biolabs) to remove theregion encoding canine p40 mature protein. The PCR product containingthe full-length canine p40 subunit (nCaIL-12 p40₉₈₇) was digested withKpn I and Nsi I restriction enzymes (available from New England Biolabs)and cloned into this digested plasmid. The coding sequence for themature canine IL-12 p40 polypeptide is encoded by SEQ ID NO:52, thecoding region for a mature (i.e. lacking a signal or leader sequence)nCaIL-12p40₉₂₁, with the complement denoted SEQ ID NO:53. Translation ofSEQ ID NO:52 yields a mature IL-12 p40 protein denoted PCaIL-12p35₃₀₇,also denoted herein as SEQ ID NO:53. nCaIL-12p40₉₂₁ was digested withSmal and XhoI restriction endonucleases (available from New EnglandBiolabs) and ligated into the pBluescript-Linker plasmid described inExample 3A digested with the same enzymes.

The resulting plasmid contains a nucleic acid molecule encoding a canineIL-12 single chain cloned at the Kpn I and Not I site into thepBluescript backbone. The complete canine IL-12 single chain insert wassequenced as described in Example 1. The nucleotide sequence of thecoding strand of nCaIL-12-single chainl₁₅₉₉is represented herein as SEQID NO:61, and its complement is denoted by SEQ ID NO:63. Translation ofthe open reading frame in SEQ ID NO:61, denoted here as nCaIL-12₁₅₉₉with a SEQ ID NO:64 suggests that canine IL-12-single chain encodes aprotein containing 533 amino acids, referred to herein as PCaIL-12₅₃₃,with an amino acid sequence denoted by SEQ ID NO:62, assuming an openreading frame in which the first codon spans from nucleotide 1 through 3of SEQ ID NO:61 and the last codon spans from nucleotide 1597 nucleotide1599 of SEQ ID NO:61. The encoded protein has a predicted molecularweight of about 59.2 kDa for the precursor protein. and about 56.8 kDafor the mature protein. The putative signal peptide cleavage site isbetween amino acid positions 22 and 23 of the canine p40 subunitprotein. Nucleic acid molecule nCaIL-12₁₅₃₃, which encodes the matureprotein contains a coding strand with SEQ ID NO:66, and a complementarystrand with SEQ ID NO:68. The amino acid sequence of the mature protein,denoted herein as pCaIL-12₅₁₁, is SEQ ID NO:67 and the mature proteinhas a predicted molecular weight of about 56.8 kDa.

Chinese hamster ovary (CHO) cells (available from ATCC, Rockville, Md.)were transiently transfected with cap40-linker-p35mature plasmid usingtechniques known to those skilled in the art and cell pellets andsupernatants were harvested after 48 hrs. Western analysis was performedon the cell pellets and supernatant samples using a polyclonal antibodyagainst human IL-12 (available from Biosource International, Camarillo,Cailf.). A faint band of the expected size (about 59.2 kDa) was detectedin the cell pellet and in the supernatant, indicating that IL-12 isproduced by this construct at detectable levels.

1-21. (canceled)
 22. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12; (b) a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 1, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74; and (c) a nucleic acid sequence fully complementary to the nucleic acid sequence of (a) or (b).
 23. The isolated nucleic acid molecule of claim 22, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:41.
 24. A recombinant virus comprising the isolated nucleic acid molecule of claim
 22. 25. A recombinant cell comprising the isolated nucleic acid molecule of claim
 22. 26. A composition comprising an excipient and the isolated nucleic acid molecule of claim
 22. 27. A kit comprising the isolated nucleic acid sequence of claim
 22. 28. An isolated nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12; (b) a nucleic acid sequence fully complementary to the nucleic acid sequence of (a).
 29. The isolated nucleic acid molecule of claim 28, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:41.
 30. A recombinant virus comprising the isolated nucleic acid molecule of claim
 28. 31. A recombinant cell comprising the isolated nucleic acid molecule of claim
 28. 32. A composition comprising an excipient and the isolated nucleic acid molecule of claim
 28. 33. A fragment of the nucleic acid molecule of claim 28, wherein said fragment is at least 70 nucleotides in length.
 34. A fragment of the nucleic acid molecule of claim 28, wherein said fragment is at least 75 nucleotides in length.
 35. A fragment of the nucleic acid molecule of claim 28, wherein said fragment is at least 100 nucleotides in length.
 36. A fragment of the nucleic acid molecule of claim 28, wherein said fragment is at least 120 nucleotides in length.
 37. A fragment of the nucleic acid molecule of claim 28, wherein said fragment is at least 200 nucleotides in length.
 38. A kit comprising the isolated nucleic acid molecule of claim
 28. 39. An isolated nucleic acid molecule consisting of a nucleic acid sequence selected from SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 or SEQ ID NO:74.
 40. A kit comprising the isolated nucleic acid molecule of claim
 39. 